TW202126284A - Lentiviral vector formulations - Google Patents

Abstract

Lentiviral vector (LV) formulations, and pharmaceutical compositions comprising such LV formulations, with improved stability and suitable for systemic administration are provided. Methods for treating disorders, especially blood disorders, using systemic administration of LV formulations are also provided.

Description

Lentiviral vector formulation

The present disclosure relates to formulations of recombinant lentiviral vectors (LV) for the treatment of diseases and related pharmaceutical products. Specifically, the present invention relates to improving the stability and quality of LV, and is also suitable for systemic and other types of administration to subjects to treat diseases (including bleeding disorders, such as hemophilia A and hemophilia B). Formulations.

Lentiviral vectors (LV) and other viral vectors are attractive tools for gene therapy (Thomas et al., 2003). LV can transduce a wide variety of tissues, including non-dividing cells such as hepatocytes, neurons, and hematopoietic stem cells. In addition, LV can integrate into the target cell genome and provide long-term gene transfer performance.

A continuing challenge in the field of gene therapy and vaccine development is to produce non-toxic liquid formulations that can keep LV structurally stable and biologically active for a longer period of time, and can withstand such as stirring, freezing /Defrost and storage conditions in a certain temperature range. It has been observed that as the freeze/thaw cycle increases and storage at higher temperatures, the LV titer decreases in a biphasic manner (Kigashikawa and Chang 2001, Virology 280, 124-131). In order to make gene therapy the most effective, it is desirable to maintain the biological activity or effectiveness of the lentiviral vector.

The biological activity of LV depends on the structural integrity of at least a closed structure consisting of: (a) the core polynucleotide, (b) the outer shell of the interconnecting capsid protein surrounding the core polynucleotide, and (c) surrounding the mutual The outer shell of the capsid protein is embedded in the lipid membrane of the glycoprotein. Unlike organic and inorganic drugs, LV is a highly complex biological structure, and small chemical or physical stressors can cause the structural integrity of the closed structure to deteriorate. Such stress sources include osmolality, buffers, pH, viscosity, electrolytes, agitation, and temperature fluctuations. The structural or structural integrity of LV is directly related to its biological activity or efficacy. Therefore, LV may lose its effectiveness due to physical instability (including denaturation, soluble and insoluble aggregation, precipitation and adsorption) and chemical instability (including hydrolysis, deamidation, and oxidation). Any of these types of degradation may result in a decrease in biological activity, and may also potentially result in the formation of by-products or derivatives with increased toxicity and/or altered immunogenicity. Therefore, a good LV formulation is not only essential to ensure a reasonable shelf life, but also essential to ensure reduced toxicity after administration to a subject (eg, via systemic administration). The search for stabilized LV to produce robust formulations (where LV is stable under a wide range of conditions) requires precise optimization of buffer type, pH, and excipients. For each set of conditions tested, different experimental methods are required to measure the stability of LV. Therefore, in view of all possible variable factors, finding the best conditions for formulating LV is challenging, and the composition of a good formulation is a priori unpredictable.

Therefore, there is a need in the industry to prepare formulations that are suitable for administration to subjects and improve the stability of LV by maintaining the quantity, structural integrity, and efficacy of LV under a certain range of conditions. Here, we disclose formulations that exhibit improved LV stability under various conditions and are suitable for systemic administration to subjects.

The present disclosure is based on the unexpected discovery that when LV is suspended in a vehicle, a lentiviral vector (LV) formulation with improved stability can be achieved, the vehicle comprising a TRIS-free buffer system (eg Phosphate or histidine buffer) in combination with carbohydrates (such as sucrose), surfactants (such as poloxamers or polysorbates), and salts (such as NaCl or other chloride salts). The contribution of surfactants (such as poloxamers) to LV stability is surprising, because surfactants are known in the industry to destabilize lipid membrane-bound particles. The following observations are also surprising: LV formulations in the pH range of about 6.0 to about 7.5 (for example, a pH of 6.5) improve LV stability, rather than making LV surface proteins (for example, capsid protein and VSV-G protein) ) Unstable and promote the disintegration or decomposition of LV. In addition, the present disclosure demonstrates that the LV formulations of the present disclosure are particularly suitable for systemic administration (eg, intravenous administration) to a subject.

In one aspect, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; ( d) surfactants; and (e) carbohydrates, wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof.

In certain embodiments, the buffer system includes phosphate buffer.

In some embodiments, the concentration of the phosphate buffer is between 5 mM and 30 mM.

In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM .

In certain embodiments, the concentration of the salt is between 80 mM and 150 mM.

In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM.

In certain embodiments, the salt is a chloride salt.

In certain embodiments, the chloride salt is NaCl.

In certain embodiments, the surfactant is a poloxamer.

In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination.

In certain embodiments, the poloxamer is poloxamer 188 (P188).

In certain embodiments, the poloxamer is poloxamer 407 (P407).

In certain embodiments, the surfactant is polysorbate.

In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

In some embodiments, the concentration of the surfactant is between 0.01% (w/v) and 0.1% (w/v).

In certain embodiments, the concentration of the surfactant is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v) .

In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v).

In certain embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v).

In certain embodiments, the carbohydrate is sucrose.

In certain embodiments, the pH of the buffer system or the formulation is between 6.0 and 8.0.

In certain embodiments, the pH is between 6.0 and 7.0.

In certain embodiments, the pH is about 6.5.

In certain embodiments, the pH is about 7.0 to about 8.0.

In certain embodiments, the pH is about 7.3.

In one aspect, the present invention relates to a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) a histidine buffer system; (c) a salt; (d) ) Surfactants; and (e) carbohydrates, wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof.

In some embodiments, the concentration of the histidine buffer is between 5 mM and 30 mM.

In certain embodiments, the concentration of the histidine buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM. mM.

In certain embodiments, the concentration of salt is between 80 mM and 150 mM.

In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM.

In certain embodiments, the salt is a chloride salt.

In certain embodiments, the chloride salt is NaCl.

In certain embodiments, the surfactant is a poloxamer.

In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination.

In certain embodiments, the poloxamer is poloxamer 188 (P188).

In certain embodiments, the poloxamer is poloxamer 407 (P407).

In certain embodiments, the surfactant is polysorbate.

In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

In some embodiments, the concentration of the surfactant is between 0.01% (w/v) and 0.1% (w/v).

In certain embodiments, the concentration of the surfactant is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v) .

In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v).

In certain embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v).

In certain embodiments, the carbohydrate is sucrose.

In certain embodiments, the pH of the buffer system or the formulation is between 6.0 and 8.0.

In certain embodiments, the pH is between 6.0 and 7.0.

In certain embodiments, the pH is about 6.5.

In certain embodiments, the pH is about 7.0 to about 8.0.

In certain embodiments, the pH is about 7.3.

In one aspect, the present invention relates to a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) a phosphate buffer system; (c) salt; (d) Surfactants; and (e) carbohydrates, wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof.

In some embodiments, the concentration of the phosphate buffer is between 5 mM and 30 mM.

In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM .

In certain embodiments, the concentration of the salt is between 80 mM and 150 mM.

In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM.

In certain embodiments, the salt is a chloride salt.

In certain embodiments, the chloride salt is NaCl.

In certain embodiments, the surfactant is a poloxamer.

In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination.

In certain embodiments, the poloxamer is poloxamer 188 (P188).

In certain embodiments, the poloxamer is poloxamer 407 (P407).

In certain embodiments, the surfactant is polysorbate.

In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

In some embodiments, the concentration of the surfactant is between 0.01% (w/v) and 0.1% (w/v).

In certain embodiments, the concentration of the surfactant is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v) .

In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v).

In certain embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v).

In certain embodiments, the carbohydrate is sucrose.

In certain embodiments, the pH of the buffer system or the formulation is between 6.0 and 8.0.

In certain embodiments, the pH is between 6.0 and 7.0.

In certain embodiments, the pH is about 6.5.

In certain embodiments, the pH is about 7.0 to about 8.0.

In certain embodiments, the pH is about 7.3.

In certain embodiments, the recombinant lentiviral vector further comprises a nucleotide sequence that is at least 80% identical to the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the recombinant lentiviral vector further comprises the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In certain embodiments, the recombinant lentiviral vector further comprises a nucleotide sequence that is at least 80% identical to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the recombinant lentiviral vector further comprises the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the recombinant lentiviral vector further comprises an enhanced transthyretin (ET) promoter.

In certain embodiments, the recombinant lentiviral vector further comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7.

In certain embodiments, the recombinant lentiviral vector is isolated from a transfected host cell selected from the group consisting of CHO cells, HEK293 cells, BHK21 cells, PER.C6 cells, NSO cells, and CAP cells.

In certain embodiments, the host cell is a CD47 positive host cell.

In one aspect, the present invention relates to a method of treating a human patient suffering from a disorder, wherein a recombinant lentiviral vector preparation is administered to the human patient, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentivirus Carrier; (b) TRIS-free buffer system; (c) salt; (d) surfactant; and (e) carbohydrate, wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the formulation is administered systemically to the human patient.

In certain embodiments, the formulation is administered intravenously.

In certain embodiments, the disorder is a bleeding disorder.

In certain embodiments, the bleeding disorder is hemophilia A or hemophilia B.

In another aspect, a recombinant lentiviral vector preparation is provided, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) Surfactants; and (e) carbohydrates, wherein the buffer system or the formulation has a pH of about 6.0 to about 7.5, and wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain exemplary embodiments, the lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof.

In certain exemplary embodiments, the buffer system includes phosphate buffer or histidine buffer. In certain exemplary embodiments, the concentration of the phosphate buffer is about 5 mM to about 30 mM. In certain exemplary embodiments, the concentration of the phosphate buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, Or about 15 mM to about 20 mM. In certain exemplary embodiments, the concentration of the histidine buffer is about 5 mM to about 30 mM. In certain exemplary embodiments, the concentration of the histidine buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM , Or about 15 mM to about 20 mM.

In certain exemplary embodiments, the concentration of the salt is about 80 mM to about 150 mM. In certain exemplary embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain exemplary embodiments, the salt is a chloride salt. In certain exemplary embodiments, the chloride salt is NaCl.

In certain exemplary embodiments, the surfactant is poloxamer. In certain exemplary embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 ( P122), Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer Sham 184 (P184), poloxamer 185 (P185), poloxamer 188 (P188), poloxamer 212 (P212), poloxamer 215 (P215), poloxamer 217 (P217) ), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334) , Poloxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 (P407) and its combination. In certain exemplary embodiments, the poloxamer is poloxamer 188 (P188). In certain exemplary embodiments, the poloxamer is poloxamer 407 (P407).

In certain exemplary embodiments, the surfactant is polysorbate. In certain exemplary embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In certain exemplary embodiments, the concentration of the surfactant is about 0.01% (w/v) to about 0.1% (w/v). In certain exemplary embodiments, the concentration of the surfactant is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v) v).

In certain exemplary embodiments, the concentration of the carbohydrate is about 0.5% (w/v) to about 5% (w/v). In certain exemplary embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v) ). In certain exemplary embodiments, the carbohydrate is sucrose.

In certain exemplary embodiments, the formulation comprises: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) about 100 mM sodium chloride; (d) about 0.05 % (W/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.3, and wherein the pharmaceutical composition is suitable for systemic administration to Human patients.

In certain exemplary embodiments, the formulation comprises: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) about 130 mM sodium chloride; (d) about 0.05 % (W/v) poloxamer 188; and (e) about 1% (w/v) sucrose, wherein the pH of the formulation is about 7.3, and wherein the pharmaceutical composition is suitable for systemic administration to Human patients.

In certain exemplary embodiments, the formulation comprises: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; (c) about 100 mM sodium chloride; (d) about 0.05% (w/v) Poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 6.5, and wherein the pharmaceutical composition is suitable for systemic administration To human patients.

In certain exemplary embodiments, the formulation comprises: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) about 100 mM sodium chloride; (d) about 0.05 % (W/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, and wherein the pharmaceutical composition is suitable for systemic administration to Human patients.

In certain exemplary embodiments, the formulation comprises: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; (c) about 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, and wherein the pharmaceutical composition is suitable for systemic administration To human patients.

In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising a factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 at least 80%, at least 85 %, at least 90%, at least 95%, or at least 99% identical nucleotide sequences. In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 80%, at least 85%, at least 90% of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3, A nucleotide sequence that is at least 95% or at least 99% identical. In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain exemplary embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain exemplary embodiments, the recombinant lentiviral vector includes an enhanced transthyretin (ET) promoter.

In certain exemplary embodiments, the recombinant lentiviral vector further comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7.

In certain exemplary embodiments, the recombinant lentiviral vector is isolated from a transfected host cell selected from the group consisting of CHO cells, HEK293 cells, BHK21 cells, PER.C6 cells, NSO cells, and CAP cells. In certain exemplary embodiments, the host cell is a CD47 positive host cell.

In another aspect, there is provided a method of treating a human patient suffering from a disorder, the method comprising administering the recombinant lentiviral vector preparation described herein to the human patient.

In certain exemplary embodiments, the formulation is administered systemically to the human patient. In certain exemplary embodiments, the formulation is administered intravenously.

In certain exemplary embodiments, the disorder is a bleeding disorder. In certain exemplary embodiments, the bleeding disorder is hemophilia A or hemophilia B.

Cross-references to related applications

This application claims the priority of U.S. Provisional Patent Application Serial No. 62/908,390 filed on September 30, 2019, the disclosure of which is hereby incorporated by reference in its entirety. Sequence Listing

This application contains a sequence listing that has been electronically filed in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on September 28, 2020 is named "709718_SA9-472PC_SeqList_ST25.txt" and has a size of 104,426 bytes.

The present disclosure particularly provides preparations (formulations) and pharmaceutical compositions of lentiviral vectors (LV) (including recombinant LV). The present disclosure also provides methods of using LV preparations to treat subjects suffering from disorders, including bleeding disorders such as hemophilia A or hemophilia B. The present disclosure further provides methods for producing LV formulations.

Generally, the terms used with regard to cell and tissue culture, molecular biology, biophysics, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the industry. Unless otherwise indicated, the methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. The enzyme reaction and purification methods are carried out according to the manufacturer's instructions, as generally done in the industry or as described herein. The terms and laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, medicine and medicinal chemistry described herein are well-known and commonly used in the industry. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

Unless otherwise defined in this article, the scientific and technical terms used in this article have the meaning generally understood by ordinary technicians in the industry. If there are any possible ambiguities, the definitions provided in this article take precedence over any dictionary or external definitions. Unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including" and other forms such as "includes" and "included" is non-limiting.

In order to make it easier to understand the present invention, first define certain terms.

As used herein, the term "vector" refers to any vehicle used to clone and/or transfer nucleic acid into a host cell. The vector can be a replicon, and another nucleic acid segment can be linked to it to realize the replication of the linked segment. "Replicon" refers to any genetic element (such as plastids, phages, cosmids, chromosomes, viruses) that functions as an autonomous replication unit in the body (that is, capable of replicating under its own control). The term "vector" includes viral vectors and non-viral vehicles used to introduce nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors are known and used in the industry, including, for example, plastids, modified eukaryotic viruses, or modified bacterial viruses. Inserting the polynucleotide into the appropriate vector can be accomplished by ligating the appropriate polynucleotide fragments into a selection vector with complementary sticky ends.

As used herein, the phrase "recombinant lentiviral vector" refers to a vector that has sufficient lentiviral genetic information to allow packaging of RNA genome into viral particles capable of infecting target cells in the presence of packaging components. Infection of target cells can include reverse transcription and integration into the target cell genome. Recombinant lentiviral vectors carry non-viral coding sequences that will be delivered from the vector to target cells. The recombinant lentiviral vector cannot replicate independently in the final target cell to produce infectious lentiviral particles. Generally, recombinant lentiviral vectors lack functional gag-pol and/or env genes and/or other genes necessary for replication. The vector of the present invention can be configured as a split-intron vector.

As used herein, the term "treatment" refers to ameliorating or alleviating one or more symptoms of a disorder. Treatment is not necessarily a cure.

As used herein, the term "human patient" refers to a human being suffering from a disease or disorder and in need of treatment for the disease or disorder.

As used herein, the phrase "systemic administration" refers to prescribing or administering a pharmaceutical composition comprising LV to the subject so that the LV is directly introduced into the blood stream of the subject. Examples of systemic administration routes include, but are not limited to, intravenous, such as intravenous injection and intravenous infusion, such as via central venous access.

As used herein, the term "about", when used in relation to a specifically recited value, means that the value can differ from the recited value by no more than 10%. For example, as used herein, the expression "about 100" includes 90 and 100 and all values in between (eg, 90, 91, 92, 93, 94, 95, etc.).

A. A formulation containing a lentiviral vector (LV) without a TRIS buffer system

In one aspect, the present invention relates to a recombinant lentiviral vector preparation comprising: (a) an effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surface Active agent; and (e) carbohydrate, wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the pH of the buffer system is about 6.0 to about 8.0. In certain embodiments, the pH of the buffer system is about 6.0 to about 7.5. In certain embodiments, the pH of the buffer system is about 6.0 to about 7.0. In certain embodiments, the pH of the buffer system is about 6.0 to about 8.0. In certain embodiments, the pH of the buffer system is about 6.5. In certain embodiments, the pH of the buffer system is about 7.3. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In certain embodiments, the concentration of the phosphate or histidine buffer is about 5 mM to about 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, or about 15 mM to about 20 mM. In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is about 80 mM to about 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In certain embodiments, the concentration of the poloxamer or polysorbate is about 0.01% (w/v) to about 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In certain embodiments, the concentration of the carbohydrate is about 0.5% (w/v) to about 5% (w/v). In certain embodiments, the chloride salt is sodium chloride (NaCl). In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In certain embodiments, the pH of the phosphate or histidine buffer is about 6.1, about 6.3, about 6.5, about 6.7, about 6.9, about 7.1, about 7.3, about 7.5, about 7.7, or about 7.9. In certain embodiments, the concentration of the phosphate or histidine buffer is about 10 mM, about 15 mM, about 20 mM, or about 25 mM. In certain embodiments, the chloride salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v). %(W/v). In certain embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.3, and The pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 130 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 1% (w/v) sucrose, wherein the pH of the formulation is about 7.3, and The pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; ( c) about 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 6.5, And wherein the pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, and The pharmaceutical composition is suitable for systemic administration to human patients.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; ( c) about 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188; and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, And wherein the pharmaceutical composition is suitable for systemic administration to human patients.

A.1. Lentiviral vector

Lentiviral vectors are part of a larger group of retroviral vectors (Coffin et al. (1997) "Retroviruses" Cold Spring Harbor Laboratory Press editor: J M Coffin, SM Hughes, HE Varmus, pages 758-763). Examples of primate lentiviruses include: human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The difference between the lentivirus family and retroviruses is that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis et al. (1992); Lewis and Emerman (1994)).

As used herein, a lentiviral vector is a vector that contains at least one component that can be derived from a lentivirus. Preferably, this component is involved in the biological mechanism of the vector infecting cells, expressing genes or replicating. In a recombinant lentiviral vector, at least a part of one or more protein coding regions necessary for replication can be removed from the virus. This makes viral vectors have replication defects. Parts of the viral genome can also be replaced by gene transfer, so that the vector can transduce the target non-dividing host cell and/or integrate its genome into the host genome.

Recombinant lentiviruses are usually pseudotyped. Pseudotyping can confer one or more advantages. For example, the env gene product of HIV-based vectors restricts these vectors to only infect cells that express a protein called CD4. However, if the env genes in these vectors have been replaced by env sequences from other RNA viruses, they can have a wider infection spectrum (Verma and Somia (1997)). The envelope glycoprotein (G) of vesicular stomatitis virus (VSV, a rhabdovirus) is an envelope protein that has been shown to pseudotype certain retroviruses. The pseudotyped VSV-G vector can be used to transduce a wide range of mammalian cells. Incorporating a non-lentiviral pseudotyped envelope (such as the VSV-G protein) has the advantage that the carrier particles can be concentrated to high titers without loss of infectivity (Akkina et al. (1996) J. Virol. 70:2581- 5). The envelope proteins of lentivirus and retrovirus obviously cannot withstand the shear force during ultracentrifugation. This may be because they are composed of two non-covalently linked subunits. The interaction between subunits may be destroyed by centrifugation. In contrast, VSV glycoprotein is composed of a single unit. Therefore, pseudotyping of VSV-G protein can provide potential advantages.

Lentiviruses include members of the bovine lentivirus group, the equine lentivirus group, the cat lentivirus group, the sheep goat lentivirus group, and the primate lentivirus group. The development of lentiviral vectors for gene therapy has been reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4:481-496. The design and use of lentiviral vectors suitable for gene therapy are described in, for example, US Patent Nos. 6,207,455 and 6,615,782. Examples of lentiviruses include but are not limited to HIV-1, HIV-2, HIV-1/HIV-2 pseudotypes, HIV-1/SIV, FIV, goat arthritis encephalitis virus (CAEV), equine infectious anemia virus and cattle Immunodeficiency virus.

In some embodiments, the lentiviral vector of the present disclosure is a "third generation" lentiviral vector. As used herein, the term "third-generation" lentiviral vector refers to a lentiviral packaging system that has the characteristics of a second-generation vector system and further lacks a functional tat gene, such as a lentiviral packaging system in which the tat gene has been deleted or inactivated system. Generally, the gene encoding rev is provided on a separate expression construct. See, for example, Dull et al. (1998) J. Virol. 72: 8463-8471. As used herein, the "second generation" lentiviral vector system refers to a lentiviral packaging system lacking functional auxiliary genes, such as a lentiviral packaging system in which the auxiliary genes vif, vpr, vpu, and nef have been deleted or inactivated. See, for example, Zufferey et al. (1997) Nat. Biotechnol. 15:871-875. As used herein, "packaging system" refers to a series of viral constructs that contain genes encoding viral proteins involved in packaging recombinant viruses. Usually, the construct of the packaging system will eventually be incorporated into the packaging cell.

In some embodiments, the third-generation lentiviral vector of the present disclosure is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotyped lentiviral vector. In some embodiments, the lentiviral vector contains a hepatocyte-specific promoter for gene transfer performance. In some embodiments, the hepatocyte-specific promoter is an enhanced transthyretin promoter. In some embodiments, the lentiviral vector contains one or more target sequences of miR-142 to reduce the immune response to the product of gene transfer. In some embodiments, the incorporation of one or more target sequences of miR-142 into the lentiviral vector of the present disclosure allows for a desired gene transfer profile. For example, the incorporation of one or more target sequences of miR-142 can inhibit gene transfer performance in intravascular and extravascular hematopoietic lineages, while maintaining gene transfer performance in non-hematopoietic cells. No tumorigenesis was detected in tumor-prone mice treated with the lentiviral vector system of the present disclosure. See Brown et al. (2007) Blood 110:4144-52; Brown et al. (2006) Nat. Ned. 12:585-91; and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

The lentiviral vectors of the present disclosure include codon-optimized polynucleotides that encode a specific protein (such as the FVIII or FIX protein described herein) for gene transfer. In one embodiment, the optimized coding sequence of the FVIII or FIX protein is operably linked to the performance control sequence. As used herein, when two nucleic acid sequences are covalently linked in a manner that allows each component nucleic acid sequence to retain its functionality, the two nucleic acid sequences are operably linked. When the coding sequence and the gene expression control sequence are covalently linked in such a way that the expression or transcription and/or translation of the coding sequence are under the influence or control of the gene expression control sequence, the coding sequence and the gene expression control sequence are said to be Operationally connected. If the induction of the promoter in the 5'gene expression sequence leads to the transcription of the coding sequence, and if the line between the two DNA sequences is not (1) leading to the introduction of frameshift mutations, (2) interfering with the promoter region to guide the coding sequence The ability to transcribe, or (3) the ability to interfere with the translation of the corresponding RNA transcript into a protein, means that the two DNA sequences are operably linked. Therefore, if the gene expression sequence can realize the transcription of the encoding nucleic acid sequence, thereby enabling the resulting transcript to be translated into the desired protein or polypeptide, the gene expression sequence will be operably linked to the encoding nucleic acid sequence.

In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting liver cells (eg, hepatocytes). Lentiviral genomes and proviral DNA usually have three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structure (matrix, capsid and nucleocapsid) protein; the pol gene encodes RNA-guided DNA polymerase (reverse transcriptase), protease and integrase; and the env gene encodes the viral envelope glycoprotein. 5'and 3'LTR are used to promote transcription and polyadenylation of virion RNA. LTR contains all other cis-acting sequences necessary for virus replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and/or SIV).

Adjacent to the 5'LTR is the sequence necessary for reverse transcription of the genome (tRNA primer binding site) and effective encapsidation of viral RNA into particles (Psi site). If the sequence necessary for encapsidation (or packaging of retroviral RNA into infectious virions) is deleted from the viral genome, the cis defect will prevent the encapsidation of genomic RNA.

However, the resulting mutant is still able to direct the synthesis of all virion proteins. The present disclosure provides a method for producing recombinant lentivirus capable of infecting non-dividing cells, the method includes transfecting a suitable vector with two or more vectors carrying packaging functions (ie, gag, pol and env, and rev and tat) Host cell. As will be disclosed below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, a first vector can provide a nucleic acid encoding a virus gag and a virus pol, and another vector can provide a nucleic acid encoding a virus env to produce packaging cells. A vector that provides a heterologous gene (identified as a transfer vector herein) is introduced into the packaging cell to produce a producer cell, which releases infectious virus particles carrying the foreign gene of interest.

According to the above-mentioned configuration of the vector and the foreign gene, the second vector can provide a nucleic acid encoding a virus envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphiphilic envelope protein, which allows the transduction of cells of humans and other species.

Examples of env genes derived from retroviruses include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Hawe murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes can also be used, such as vesicular stomatitis virus (VSV) protein G (VSV-G), hepatitis virus env gene, and influenza virus env gene. In some embodiments, the viral env nucleic acid sequence is operably associated with regulatory sequences described elsewhere herein. In certain embodiments, the formulation buffer of the present disclosure imparts stability to the lentivirus and provides long-term frozen storage, especially for lentivirus containing VSV-G. The formulation buffer of the present invention provides enhanced lentivirus stability when freezing and thawing and exposure to elevated temperatures, especially for lentivirus containing VSV-G.

In certain embodiments, the lentiviral vector deletes the HIV virulence genes env, vif, vpr, vpu, and nef without compromising the vector's ability to transduce non-dividing cells. In some embodiments, the lentiviral vector contains a deletion of the U3 region of the 3'LTR. The deletion of the U3 region can be a complete deletion or a partial deletion.

In some embodiments, the lentiviral vector of the present disclosure containing the FVIII nucleotide sequence described herein can be used for down-transfection in cells: (a) the first nucleus including gag, pol, or gag and pol genes The nucleotide sequence and (b) include the second nucleotide sequence of the heterologous env gene; wherein the lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence including the rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

In certain embodiments, the lentiviral vector of the present disclosure includes one or more nucleotide sequences encoding gag protein, Rev response element, central polypurine region (cPPT), or any combination thereof.

In some embodiments, the lentiviral vector displays on its surface one or more polypeptides that improve the targeting and/or activity of the lentiviral vector or the encoded FVIII polypeptide. The one or more polypeptides can be encoded by the lentiviral vector, or can be incorporated during budding of the lentiviral vector from the host cell. During the production of the lentivirus, viral particles bud out from the production host cell. During the budding process, the virus particles are provided with a lipid coating, which is derived from the lipid coating of the host cell. Therefore, the lipid coating of the viral particle may include a membrane-bound polypeptide previously present on the surface of the host cell.

In some embodiments, the lentiviral vector displays one or more polypeptides on its surface, and the one or more polypeptides inhibit the immune response to the lentiviral vector after being administered to a human subject. In some embodiments, the surface of the lentiviral vector contains one or more CD47 molecules. CD47 is a "self-marker" protein, which is ubiquitously expressed on human cells. The surface expression of CD47 inhibits the phagocytosis of endogenous cells induced by macrophages through the interaction of CD47 and SIRPα expressed by macrophages. Cells expressing high levels of CD47 are unlikely to be targeted and destroyed by human macrophages in vivo.

In some embodiments, the lentiviral vector contains a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is produced in a cell line with a high expression level of CD47. In certain embodiments, the lentiviral vector ^{is produced in CD47 high} cells, wherein the cells have high CD47 expression on the cell membrane. In a specific embodiment, the lentiviral vector ^{is produced in CD47-high} HEK 293T cells, where HEK 293T has a high CD47 expression on the cell membrane. In some embodiments, HEK 293T cells are modified to have increased CD47 performance relative to unmodified HEK 293T cells. In certain embodiments, CD47 is human CD47.

In some embodiments, the lentiviral vector has little or no surface manifestation of major histocompatibility complex class I (MHC-I). The surface MHC-I displays peptide fragments from "non-self" proteins in cells, such as protein fragments that indicate infection, thereby promoting immune responses against cells. In some embodiments, the lentiviral vector ^{is produced in MHC-I low} cells, wherein the cells have reduced MHC-I expression on the cell membrane. In some embodiments, the lentivirus vector MHC-I ^- (or ^"non-MHC-I", "MHC-1 ^negative" or "negative MHC-") produced in the cells, wherein said cells lack MHC-I expression .

In a specific embodiment, the lentiviral vector comprises a lipid coating that contains a high concentration of CD47 polypeptide and lacks MHC-I polypeptide. In certain embodiments, the lentiviral vector ^{is produced in a CD47 high} /MHC-I ^low cell line, such as a CD47 ^high /MHC-I ^low HEK 293T cell line. In some embodiments, the lentiviral vector ^{is produced in a CD47 high} /MHC-I ^cell-free line, such as a CD47 ^high /MHC-I ^HEK-free 293T cell line.

Examples of lentiviral vectors are disclosed in U.S. Patent No. 9,050,269 and International Publication Nos. WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, which are incorporated herein by reference in their entireties.

In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a nucleotide sequence including the nucleotide sequence shown in Table 1.

Table 1: Sequence SEQ ID NO describe sequence 1 FVIII coding sequence 2 FVIII coding sequence containing XTEN (XTEN is in bold and underlined) ACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGCACAAGTGAGTCTGCAACTCCCGAGTCCGGACCTGGCTCCGAGCCTGCCACTAGCGGCTCCGAGACTCCGGGAACTTCCGAGAGCGCTACACCAGAAAGCGGACCCGGAACCAGTACCGAACCTAGCGAGGGCTCTGCTCCGGGCAGCCCAGCCGGCTCTCCTACATCCACGGAGGAGGGCACTTCCGAATCCGCCACCCCGGAGTCAGGGCCAGGATCTGAACCCGCTACCTCAGGCAGTGAGACGCCAGGAACGAGCGAGTCCGCTACACCGGAGAGTGGGCCAGGGAGCCCTGCTGGATCTCCTACGTCCACTGAGGAAGGGTCACCAGCGGGCTCGCCCACCAGCACTGAAGAAGGTGCCTCGAGC 3 FIX-R338L coding sequence ( signal peptide in bold and underlined) ATGCAGAGAGTCAACATGATTATGGCTGAGTCACCTGGGCTGATTACTATTTGCCTGCTGGGCTACCTGCTGTCCGCCGAGTGTACCGTGTTCCTGGACCATGAGAACGCAAATAAGATCCTGAACAGGCCCAAAAGA 4 Mature FVIII peptide 5 FVIII amino acid sequence containing XTEN (XTEN is in bold and underlined) TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGASS 6 FIX-R338L amino acid sequence ( signal peptide in bold and underlined) MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKR YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLLSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT 7 miR-142 target sequence tccataaagtaggaaacactaca

When administered at a dose of 5× ^{10 10} TU/kg or lower, 10 ⁹ TU/kg or lower, or 10 ⁸ TU/kg or lower, the lentiviral vector of the present disclosure is therapeutically effective. At such doses, regarding the basic level in the subject, relative to the level in the subject administered the control lentiviral vector, relative to the level in the subject administered the control nucleic acid molecule, or relative At the level of subjects after administration of the polypeptide encoded by the control nucleic acid molecule, the administration of the lentiviral vector of the present disclosure can cause the plasma FVIII activity in subjects in need to increase at least about 2-fold, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least about 100 times, at least about 110, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times.

In certain embodiments, it is useful to include one or more miRNA target sequences within the lentiviral vector, the miRNA target sequences being operably linked, for example, to gene translocation, such as optimized FVIII gene translocation. Therefore, the present disclosure also provides at least one miRNA sequence target that is operably linked to the optimized FVIII or optimized FIX nucleotide sequence or otherwise inserted into the lentiviral vector. The inclusion of more than one copy of the miRNA target sequence in the lentiviral vector can increase the effectiveness of the system.

It also includes different miRNA target sequences. For example, a lentiviral vector that exhibits more than one gene transfer may have gene transfer under the control of more than one miRNA target sequence that may be the same or different. The miRNA target sequence can be tandem, but other arrangements are also included. Gene transfer expression cassettes containing miRNA target sequences can also be inserted into lentiviral vectors in antisense orientation. Antisense targeting can be used to produce viral particles to avoid the expression of gene products that might otherwise be toxic to the production cell.

In other embodiments, the lentiviral vector contains 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence. In certain embodiments, the lentiviral vector does not include any miRNA target sequences. The choice of whether to include miRNA target sequence (and quantity) will be guided by known parameters such as planned tissue target, required performance level, etc.

In one example, the target sequence is the miR-223 target, which has been reported to be the most effective in blocking performance in bone marrow-directed progenitor cells, and at least partially blocking performance in more primitive HSPCs. The miR-223 target can block the expression in differentiated myeloid cells (including granulocytes, monocytes, macrophages, myeloid dendritic cells). The miR-223 target may also be suitable for gene therapy applications that rely on robust gene transfer performance in the lymphoid or red blood cell lineage. The miR-223 target can also block performance very effectively in human HSCs.

In another embodiment, the target sequence is the miR142 target (tccataaagtaggaaacactaca (SEQ ID NO: 7)). In one embodiment, the lentiviral vector contains 4 copies of miR-142 target sequence. In some embodiments, the complementary sequence of hematopoietic-specific microRNA (such as miR-142 (142T)) is incorporated into the 3'untranslated region of the lentiviral vector, so that the transcripts encoding gene transfer are prone to miRNA-mediated Guided downward adjustment. In this way, gene translocation performance can be prevented in hematopoietic lineage antigen presenting cells (APC), while maintaining the gene translocation performance in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose strict post-transcriptional control on gene transfer performance, and thus enable stable delivery and long-term performance of gene transfer. In some embodiments, miR-142 regulation prevents immune-mediated clearance of transduced cells and/or induces antigen-specific regulatory T cells (T regs), and mediates robust immune tolerance to gene-transformed antigens. by.

In some embodiments, the target sequence is the miR181 target. Chen C-Z and Lodish H, Seminars in Immunology (2005) 17(2):155-165 disclosed miR-181, which is a miRNA specifically expressed in B cells in the bone marrow of mice (Chen and Lodish, 2005). The document also discloses that some human miRNAs are related to leukemia.

The target sequence can be fully or partially complementary to the miRNA. The term "fully complementary" means that the nucleic acid sequence of the target sequence is 100% complementary to the sequence of the miRNA that recognizes the target sequence. The term "partially complementary" means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes the target sequence, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, the partially complementary target sequence effectively recognizes the corresponding miRNA, and achieves the prevention or reduction of gene translocation performance in the cell expressing the miRNA. Examples of miRNA target sequences are described in the following documents: WO 2007/000668, WO 2004/094642, WO 2010/055413 or WO 2010/125471, which are incorporated herein by reference in their entirety.

A.2. Excipients, carriers and other ingredients of the formulation

For the purpose of gene therapy, lentiviral vectors (LV) are usually administered systemically, that is, directly into the patient's bloodstream. Therefore, it is of broad significance to produce LV formulations that are non-toxic but still maintain the stability and efficacy of LV. When testing vehicles to produce LV formulations suitable for systemic administration, certain core principles must be kept in mind. In order to ensure the smallest impact on the subject administering the formulation, the pH, ion concentration, and volumetric osmolality must be optimized to match the physiological conditions. The combination of buffer, salt, and carbohydrate (respectively used to adjust pH, ion concentration, and volume osmolality) is not a priori determinable and must be tested experimentally.

For example, US20170073702A1 discloses TSSM vehicle (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3), which may have been predicted to be effective in mammals Systemic administration is non-toxic. However, it was found (see Example 2) that formulations using TSSM alone or in combination with LV are toxic to mice. However, surprisingly, when using a phosphate vehicle (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3) or histidine vehicle (20 mM histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 6.5), the formulation is non-toxic to mice.

Because it cannot be predicted a priori, the formulation with a phosphate vehicle resulted in higher stability and integrity of LV compared to the TSSM formulation (Example 3 and Example 4). Unexpectedly, the histidine vehicle imparts greater stability to the LV than the phosphate vehicle (Example 4). This key feature of histidine formulations is that compared with the neutral pH (pH 7.3) of phosphate or TRIS buffer, the compatibility of the lentiviral vector with the vehicle’s lower pH (pH 6.5) is at least better than that of neutral pH (pH 7.3). It is surprising for the following reasons. The VSV-G envelope protein is an important component of the lentiviral vector, which is beneficial to the infectivity of the lentiviral vector to cells. The pI (isoelectric point) of VSV-G is about 5, which means that as the pH of the solution approaches the pI, the charge on the protein becomes more neutral. As the charges of proteins become more neutral, they are more capable of attracting each other, and this can lead to aggregation (deterioration mechanism), thereby losing the ability to infect.

It was also surprising that the inclusion of the surfactant poloxamer 188 (P188) in the phosphate and histidine formulations imparted increased LV stability and integrity compared to the TSSM formulations. This was unexpected because it is predicted that the surfactant will destabilize the outer lipid membrane envelope of the LV, thereby reducing the stability and integrity of the LV. Similarly, it would not be obvious a priori to remove mannitol from the formulation to produce a formulation with higher LV stability and integrity (Example 3 and Example 4).

Thus, based on the findings described herein, it was found that a lentiviral vector formulation containing a TRIS-free buffer system provides increased lentiviral vector stability and integrity. Those familiar with the art will understand that a TRIS-free buffer system refers to any buffer system that does not contain TRIS (also known as tris(hydroxymethyl)aminomethane, trimethylamine, or THAM). In certain embodiments, the TRIS-free buffer system includes phosphate. In certain embodiments, the TRIS-free buffer system contains histidine.

In certain embodiments, the pH of the TRIS-free buffer system or formulation is about 6.0 to about 8.0. In certain embodiments, the pH of the TRIS-free buffer system or formulation is from about 6.0 to about 7.5. In certain embodiments, the pH of the TRIS-free buffer system or formulation is about 6.0 to about 7.0. In certain embodiments, the pH of the TRIS-free buffer system or formulation is from about 7.0 to about 8.0. In certain embodiments, the pH of the TRIS-free buffer system or formulation is about 6.5. In certain embodiments, the pH of the TRIS-free buffer system or formulation is about 7.3.

Those skilled in the art will be able to determine the appropriate buffer components to be used in the TRIS-free buffer system of the formulations disclosed herein to maintain the target pH or target pH range. In certain embodiments, the TRIS-free buffer system includes a buffer component with an effective pH buffering range of about 6.0 to about 8.0. In certain embodiments, the TRIS-free buffer system includes a buffer component with an effective pH buffering range of about 6.0 to about 7.5. In certain embodiments, the TRIS-free buffer system includes a buffer component with an effective pH buffering range of about 6.0 to about 7.0. In certain embodiments, the TRIS-free buffer system includes a buffer component with an effective pH buffering range of about 7.0 to about 8.0. In certain embodiments, the TRIS-free buffer system includes a buffer component with an effective pH buffering range that can maintain a pH of 6.5. In certain embodiments, the TRIS-free buffer system includes a buffer component that has an effective pH buffering range that can maintain a pH of 7.3.

The composition comprising the lentiviral gene therapy vector disclosed herein or the host cell of the present disclosure (eg, liver cells targeted with the lentiviral gene therapy vector disclosed herein) may contain a suitable pharmaceutically acceptable carrier. For example, the composition may contain excipients and/or auxiliaries that facilitate processing of the active compound into a formulation designed for delivery to the site of action.

The pharmaceutical composition can be formulated for parenteral administration (ie, intravenous, subcutaneous, or intramuscular) by bolus injection. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. The composition may take a form such as the following: a suspension, solution or emulsion in an oily or aqueous vehicle, and contains formulation agents such as suspending agents, stabilizers and/or dispersants. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (for example, pyrogen-free water).

Formulations suitable for parenteral administration also include aqueous solutions of the active compounds in water-soluble form (eg, water-soluble salts). In addition, a suspension of the active compound may be administered as an appropriate oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils (for example, sesame oil) or synthetic fatty acid esters (for example, ethyl oleate or triglycerides). Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate molecules of the present disclosure for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, Glycerin, ethanol, etc. In some embodiments, the composition includes isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride. In other embodiments, the composition contains pharmaceutically acceptable substances (such as wetting agents) or minor amounts of auxiliary substances (such as wetting or emulsifying agents, preservatives or buffers) that enhance the shelf life or effectiveness of the active ingredients.

The compositions of the present disclosure may be in a variety of forms, including, for example, liquids (eg, injectable and infusible solutions), dispersions, suspensions, semi-solid, and solid dosage forms. The preferred form depends on the mode of administration and therapeutic application.

The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent having one or a combination of the ingredients listed above as necessary, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from the previously sterile filtered solution . The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as monostearate and gelatin.

The active ingredient can be formulated with a controlled release formulation or device. Examples of such formulations and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods of preparing such formulations and devices are known in the industry. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J. R. Robinson, Marcel Dekker, Inc., New York, 1978.

Injectable long-acting formulations can be prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the polymer used, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Injectable long-acting formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into the composition. In one embodiment, another coagulation factor or a variant, fragment, analog or derivative thereof is used to formulate the chimeric protein of the present disclosure. For example, coagulation factors include but are not limited to factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue Factor (rsTF) or any of the preceding forms of activation. The coagulation factor or hemostatic agent may also include anti-fibrinolytic drugs, such as epsilon-aminocaproic acid, tranexamic acid.

The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be reduced or increased proportionally as shown by the urgency of the treatment situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pennsylvania 1980).

In addition to the active compound, the liquid dosage form can also contain inert ingredients, such as water, ethanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, Fatty acid esters of oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan.

Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E. W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, Glycerin, propylene, glycol, water, ethanol, etc. The composition may also contain pH buffering agents and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition may take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid, such as a syrup or suspension. Liquids may include suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifiers (lecithin or gum arabic), non-aqueous vehicles (for example, almond oil, oily esters, ethanol or fractionated vegetable oils) ) And preservatives (for example, methyl paraben or propyl paraben or sorbic acid). The formulation may also include flavoring agents, coloring agents and sweetening agents. Alternatively, the composition may be presented as a dry product for constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.

For administration by inhalation, the compounds used in accordance with the present disclosure are conveniently in the form of an aerosolized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack optionally with a propellant or A nebulizer is delivered, and the propellant is, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges of gelatin used in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition may also be formulated for rectal administration as, for example, a suppository containing a conventional suppository base (such as cocoa butter or other glycerides) or a retention enemas.

In one embodiment, the pharmaceutical composition comprises a lentiviral vector and a pharmaceutically acceptable carrier, the lentiviral vector comprising an optimized nucleic acid molecule encoding a polypeptide having factor VIII or factor IX activity. In another embodiment, the pharmaceutical composition comprises a host cell (for example, a liver cell) and a pharmaceutically acceptable carrier, the host cell comprises a lentiviral vector, and the lentiviral vector comprises a gene encoding a factor VIII or factor IX. Optimized nucleic acid molecules for active polypeptides.

In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. Parenteral administration can be intravenous or subcutaneous administration.

The fundamental aspect that ensures the transformation of therapeutic formulations from the laboratory to a manufacturable and marketable product of high and consistent quality is their stability in the dosage form. Due to its complex chemistry and structure, proteins (such as virus surface proteins and capsid proteins) are prone to various forms of physical and chemical degradation, which may impair the biological efficacy and safety of the final drug product. For example, protein aggregation is a key quality attribute that is regularly monitored for protein-based products and is critical for determining the shelf life of the product. Fundamentally, protein aggregation is associated with the stability of the protein's native form, and its growth in non-native cells (such as non-native mammalian cells) is usually associated with increased aggregation rate and extent. Therefore, it is not surprising that attempts to control and minimize aggregation (kinetic stability) during product shelf life are often mediated through the use of excipients or formulation conditions aimed at increasing the conformational stability of the protein. Essentially, the goal is to stabilize the protein in its natural conformation so as to minimize the population of "unnatural" species that have the ability to aggregate. Sugars and polyols (such as sucrose, trehalose, mannitol, sorbitol, etc.) are usually used to stabilize the protein in its natural state and reduce the aggregation rate. However, the undesirable effect of using these stabilizers is a concentration-dependent increase in solution viscosity.

Solution viscosity is a key attribute of protein products (especially those formulated at high protein concentrations), and it can significantly affect the utility and success of the product. The manufacturability of the product and the end use of the patient or healthcare practitioner are closely related to the ability of the solution to flow seamlessly. For example, high viscosity may require the use of a dedicated dosing device or program, which may not always be suitable for the desired population, thereby limiting the use of the product. In other cases, high solution viscosity may require the application of manufacturing techniques (such as high temperature treatment) that may negatively affect the stability of the protein. Therefore, viscosity reducing excipients (such as salts and amino acids) are usually used in solutions with high protein concentrations. However, these excipients may have a negative impact on the stability of the protein, resulting in an increase in the aggregation rate of the solution compared to a high viscosity control solution lacking a viscosity reducer. Essentially, commonly used stabilizers and viscosity reducing excipients may have opposite effects on product performance, thereby complicating product development.

Another key attribute of injectable products (most protein-based products) to consider is their osmolality. Although intravenous solutions usually need to be isotonic, subcutaneous solutions are usually hypertonic. In fact, there is evidence in the literature that after subcutaneous administration, hypertonic formulations produce enhanced protein bioavailability (Fathallah, AM et al ., Biopharm Drug Dispos. 2015, March; 36(2):115-25) . Therefore, during the clinical development phase, it is necessary to carefully monitor and characterize the influence of the solution weight osmolality (and therefore tonicity) on the discomfort and/or response of the injection site and the bioavailability in the patient population.

Formulations can sometimes contain surfactants (such as poloxamers and polysorbates) that may impart certain benefits. Poloxamer is a nonionic poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) copolymer. They are used as surfactants, emulsifiers, solubilizers, dispersants and in vivo absorption enhancers in pharmaceutical formulations. Poloxamer is a synthetic triblock copolymer with the following core formula: (PEO)a-(PPO)b-(PEO)a. All poloxamers have a similar chemical structure, but their molecular weight and the composition of the hydrophilic PEO block and the hydrophobic PPO block are different. The two most commonly used poloxamers are Poloxamer 188 (a = 80, b = 27) with a molecular weight range of 7680 to 9510 Da and Poloxamer 407 (a = 101) with a molecular weight range of 9840 to 14600 Da , B = 56). Other poloxamers include: poloxamer 101 (P101), poloxamer 105 (P105), poloxamer 108 (P108), poloxamer 122 (P122), poloxamer 123 (P123) ), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer Poloxamer 185 (P185), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234) , Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402) and Poloxamer 403 (P403).

Polysorbates are a type of emulsifier used in some pharmaceutical and food preparation formulations. Polysorbate is an oily liquid derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Common brand names of polysorbate include Scatics, Alkest, Canarcel, and Tween. The naming convention of polysorbate is usually as follows: polysorbate x (polyoxyethylene (y) sorbitan mono'z'), where x is associated with the fatty acid of polyoxyethylene sorbitan (z) The type is related, and y refers to the total number of _{oxyethylene-(CH 2} CH _{2 O)- groups found in the polysorbate molecule.} Examples of polysorbates include: (a) polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), (b) polysorbate 40 (polyoxyethylene (20) sorbitan Alcohol monopalmitate), (c) polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and (d) polysorbate 80 (polyoxyethylene (20) sorbitan Sugar alcohol monooleate).

B. Lentiviral vector (LV) formulations for the treatment of blood disorders

In one aspect, the present invention relates to a recombinant lentiviral vector preparation comprising: (a) an effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surface Active agent; (e) carbohydrate, and (f) with the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 A nucleotide sequence that is at least 80% identical in sequence, wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the vector includes the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the vector includes the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In another aspect, the present invention provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; ( d) surfactants; and (e) carbohydrates, wherein the recombinant lentiviral vector comprises a nucleic acid comprising at least the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical nucleotide sequence or at least 80%, at least 85%, at least the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 The nucleotide sequence is 90%, at least 95%, or at least 99% identical, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the pH of the buffer system is about 6.0 to about 8.0. In certain embodiments, the pH of the buffer system is about 6.0 to about 7.5. In certain embodiments, the pH of the buffer system is about 6.0 to about 7.0. In certain embodiments, the pH of the buffer system is about 6.0 to about 8.0. In certain embodiments, the pH of the buffer system is about 6.5. In certain embodiments, the pH of the buffer system is about 7.3. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In certain embodiments, the concentration of the phosphate or histidine buffer is about 5 mM to about 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, or about 15 mM to about 20 mM. In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is about 80 mM to about 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In certain embodiments, the concentration of the poloxamer or polysorbate is about 0.01% (w/v) to about 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In certain embodiments, the concentration of the carbohydrate is about 0.5% (w/v) to about 5% (w/v). In certain embodiments, the chloride salt is sodium chloride (NaCl). In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In certain embodiments, the pH of the phosphate or histidine buffer is about 6.1, about 6.3, about 6.5, about 6.7, about 6.9, about 7.1, about 7.3, about 7.5, about 7.7, or about 7.9. In certain embodiments, the concentration of the phosphate or histidine buffer is about 10 mM, about 15 mM, about 20 mM, or about 25 mM. In certain embodiments, the chloride salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (w/v). %(W/v). In certain embodiments, the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188 and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.3, wherein The pharmaceutical composition is suitable for systemic administration to human patients, and wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises factor VIII (FVIII) shown in SEQ ID NO: 1 or SEQ ID NO: 2. The coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence, or at least 80%, at least at least 80% to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 85%, at least 90%, at least 95%, or at least 99% identical nucleotide sequence, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 130 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188 and (e) about 1% (w/v) sucrose, wherein the pH of the formulation is about 7.3, wherein The pharmaceutical composition is suitable for systemic administration to human patients, and wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises factor VIII (FVIII) shown in SEQ ID NO: 1 or SEQ ID NO: 2. The coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence, or at least 80%, at least at least 80% to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 85%, at least 90%, at least 95%, or at least 99% identical nucleotide sequence, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; ( c) about 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188 and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 6.5, Wherein the pharmaceutical composition is suitable for systemic administration to human patients, and where the recombinant lentiviral vector comprises a nucleic acid comprising a factor VIII (FVIII) shown in SEQ ID NO: 1 or SEQ ID NO: 2 ) The coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence, or at least 80% to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3, The nucleotide sequence is at least 85%, at least 90%, at least 95%, or at least 99% identical, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 10 mM phosphate; (c) ) About 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188 and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, wherein The pharmaceutical composition is suitable for systemic administration to human patients, and wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises factor VIII (FVIII) shown in SEQ ID NO: 1 or SEQ ID NO: 2. The coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence, or at least 80%, at least at least 80% to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 85%, at least 90%, at least 95%, or at least 99% identical nucleotide sequence, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the present disclosure provides a recombinant lentiviral vector preparation, the recombinant lentiviral vector preparation comprising: (a) a therapeutically effective dose of a recombinant lentiviral vector; (b) about 20 mM histidine; ( c) about 100 mM sodium chloride; (d) about 0.05% (w/v) poloxamer 188 and (e) about 3% (w/v) sucrose, wherein the pH of the formulation is about 7.0, Wherein the pharmaceutical composition is suitable for systemic administration to human patients, and where the recombinant lentiviral vector comprises a nucleic acid comprising a factor VIII (FVIII) shown in SEQ ID NO: 1 or SEQ ID NO: 2 ) The coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence, or at least 80% to the factor IX (FIX) coding sequence shown in SEQ ID NO: 3, The nucleotide sequence is at least 85%, at least 90%, at least 95%, or at least 99% identical, and wherein the pharmaceutical composition is suitable for systemic administration to a human patient. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid comprising the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid consisting of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the recombinant lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the recombinant lentiviral vector comprises an enhanced transthyretin (ET) promoter. In certain embodiments, the recombinant lentiviral vector comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7.

In certain embodiments, the recombinant lentiviral vector is isolated from transfected host cells including CHO cells, HEK293 cells, BHK21 cells, PER.C6 cells, NSO cells, and CAP cells. In certain embodiments, the host cell is a CD47 positive host cell.

In certain embodiments, the formulation is administered systemically to the human patient. In certain embodiments, the formulation is administered intravenously.

In certain embodiments, the pH of the buffer system is between 6.0 and 8.0. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In some embodiments, the concentration of the phosphate or histidine buffer is between 5 mM and 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM . In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is between 80 mM and 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In some embodiments, the concentration of the poloxamer or polysorbate is between 0.01% (w/v) and 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v). In certain embodiments, the chloride salt is NaCl. In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the pH of the phosphate or histidine buffer is 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9. In some embodiments, the concentration of the phosphate or histidine buffer is 10 mM, 15 mM, 20 mM or 25 mM. In certain embodiments, the chloride salt is 100 mM, 110 mM, 130 mM, or 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is 0.03% (w/v), 0.05% (w/v), 0.07% (w/v) or 0.09% (w/v) v). In some embodiments, the carbohydrate concentration is 1% (w/v), 2% (w/v), 3% (w/v), or 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

In one aspect, the present invention relates to a recombinant lentiviral vector preparation comprising: (a) an effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surface Active agent; (e) carbohydrate, and (f) enhanced transthyretin (ET) promoter, wherein the pharmaceutical composition is suitable for systemic administration to human patients. In certain embodiments, the vector further comprises a factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a factor IX (FIX) coding sequence shown in SEQ ID NO: 3 The nucleotide sequence is at least 80% identical, wherein the pharmaceutical composition is suitable for systemic administration to human patients. In certain embodiments, the vector includes the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the vector includes the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the vector further comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the pH of the buffer system is between 6.0 and 8.0. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In some embodiments, the concentration of the phosphate or histidine buffer is between 5 mM and 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM . In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is between 80 mM and 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In some embodiments, the concentration of the poloxamer or polysorbate is between 0.01% (w/v) and 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v). In certain embodiments, the chloride salt is NaCl. In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the pH of the phosphate or histidine buffer is 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9. In some embodiments, the concentration of the phosphate or histidine buffer is 10 mM, 15 mM, 20 mM or 25 mM. In certain embodiments, the chloride salt is 100 mM, 110 mM, 130 mM, or 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is 0.03% (w/v), 0.05% (w/v), 0.07% (w/v) or 0.09% (w/v) v). In some embodiments, the carbohydrate concentration is 1% (w/v), 2% (w/v), 3% (w/v), or 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407). In one aspect, the present invention relates to a recombinant lentiviral vector preparation comprising: (a) an effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surface Active agent; (e) carbohydrate; and (f) a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7, wherein the pharmaceutical composition is suitable for systemic administration To human patients. In certain embodiments, the vector further comprises an enhanced transthyretin (ET) promoter. In certain embodiments, the vector further comprises a factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a factor IX (FIX) coding sequence shown in SEQ ID NO: 3 At least 80% identical nucleotide sequence. In certain embodiments, the vector includes the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the vector includes the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the vector further comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the pH of the buffer system is between 6.0 and 8.0. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In some embodiments, the concentration of the phosphate or histidine buffer is between 5 mM and 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM . In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is between 80 mM and 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In some embodiments, the concentration of the poloxamer or polysorbate is between 0.01% (w/v) and 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v). In certain embodiments, the chloride salt is NaCl. In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the pH of the phosphate or histidine buffer is 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9. In some embodiments, the concentration of the phosphate or histidine buffer is 10 mM, 15 mM, 20 mM or 25 mM. In certain embodiments, the chloride salt is 100 mM, 110 mM, 130 mM, or 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is 0.03% (w/v), 0.05% (w/v), 0.07% (w/v) or 0.09% (w/v) v). In some embodiments, the carbohydrate concentration is 1% (w/v), 2% (w/v), 3% (w/v), or 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

In one aspect, the present invention relates to a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector is isolated from a transfected host cell including: CHO cells, HEK293 cells, BHK21 cells, PER.C6 cells, NSO cells, and CAP cells , Wherein the recombinant lentiviral vector preparation includes: (a) an effective dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surfactant; and (e) carbohydrate, wherein The pharmaceutical composition is suitable for systemic administration to human patients. In certain embodiments, the host cell is a CD47 positive host cell. In certain embodiments, the vector further comprises an enhanced transthyretin (ET) promoter. In certain embodiments, the vector further comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7. In certain embodiments, the vector further comprises a factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a factor IX (FIX) coding sequence shown in SEQ ID NO: 3 At least 80% identical nucleotide sequence. In certain embodiments, the vector includes the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the vector includes the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the vector further comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the pH of the buffer system is between 6.0 and 8.0. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In some embodiments, the concentration of the phosphate or histidine buffer is between 5 mM and 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM . In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is between 80 mM and 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In some embodiments, the concentration of the poloxamer or polysorbate is between 0.01% (w/v) and 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v). In certain embodiments, the chloride salt is NaCl. In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the pH of the phosphate or histidine buffer is 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9. In some embodiments, the concentration of the phosphate or histidine buffer is 10 mM, 15 mM, 20 mM or 25 mM. In certain embodiments, the chloride salt is 100 mM, 110 mM, 130 mM, or 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is 0.03% (w/v), 0.05% (w/v), 0.07% (w/v) or 0.09% (w/v) v). In some embodiments, the carbohydrate concentration is 1% (w/v), 2% (w/v), 3% (w/v), or 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

In one aspect, the present invention relates to a method of treating a human patient suffering from a disorder, wherein a recombinant lentiviral vector preparation is administered to the human patient systemically, the recombinant lentiviral vector preparation comprising: (a) (a) effective Dose of recombinant lentiviral vector; (b) no TRIS buffer system; (c) salt; (d) surfactant; and (e) carbohydrate, wherein the pharmaceutical composition is suitable for systemic administration to human patients. In certain embodiments, the formulation is administered systemically to the human patient. In certain embodiments, the formulation is administered intravenously.

In certain embodiments, the disorder is a bleeding disorder. In certain embodiments, the bleeding disorder is hemophilia A or hemophilia B.

In certain embodiments, the vector further comprises an enhanced transthyretin (ET) promoter. In certain embodiments, the vector further comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7. In certain embodiments, the vector further comprises a factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a factor IX (FIX) coding sequence shown in SEQ ID NO: 3 At least 80% identical nucleotide sequence. In certain embodiments, the vector includes the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the vector includes the factor IX (FIX) coding sequence shown in SEQ ID NO: 3.

In certain embodiments, the vector further comprises a nucleotide sequence encoding VSV-G or a fragment thereof. In certain embodiments, the pH of the buffer system is between 6.0 and 8.0. In some embodiments, the buffer system is phosphate buffer or histidine buffer. In some embodiments, the concentration of the phosphate or histidine buffer is between 5 mM and 30 mM. In certain embodiments, the concentration of the phosphate buffer is about 10 to about 20 mM, about 10 to about 15 mM, about 20 to about 30 mM, about 20 to about 25 mM, or about 15 to about 20 mM . In certain embodiments, the salt is a chloride salt. In certain embodiments, the concentration of the chloride salt is between 80 mM and 150 mM. In certain embodiments, the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. In certain embodiments, the surfactant is poloxamer or polysorbate. In some embodiments, the concentration of the poloxamer or polysorbate is between 0.01% (w/v) and 0.1% (w/v). In certain embodiments, the carbohydrate is sucrose. In some embodiments, the concentration of the carbohydrate is between 0.5% (w/v) and 5% (w/v). In certain embodiments, the chloride salt is NaCl. In certain embodiments, the poloxamer is selected from Poloxamer 101 (P101), Poloxamer 105 (P105), Poloxamer 108 (P108), Poloxamer 122 (P122) , Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215), Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer Loxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 ( P407) and its combination. In certain embodiments, the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the pH of the phosphate or histidine buffer is 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9. In some embodiments, the concentration of the phosphate or histidine buffer is 10 mM, 15 mM, 20 mM or 25 mM. In certain embodiments, the chloride salt is 100 mM, 110 mM, 130 mM, or 150 mM. In certain embodiments, the concentration of the poloxamer or polysorbate is 0.03% (w/v), 0.05% (w/v), 0.07% (w/v) or 0.09% (w/v) v). In some embodiments, the carbohydrate concentration is 1% (w/v), 2% (w/v), 3% (w/v), or 4% (w/v). In certain embodiments, the poloxamer is poloxamer 188 (P188). In certain embodiments, the poloxamer is poloxamer 407 (P407).

B.1. Bleeding disorders

Bleeding disorders are caused by impaired blood's ability to form clots at the site of blood vessel damage. There are several types of bleeding disorders, including hemophilia A, hemophilia B, von Willebrand disease, and rare factor deficiencies. Hemophilia A is caused by a factor VIII mutation or insufficient expression of factor VIII (FVIII), while hemophilia B is caused by a factor IX mutation or insufficient expression of factor IX (FIX). .

According to the US Centers for Disease Control and Prevention, about 1 in 5,000 live births has hemophilia. In the United States, about 20,000 people suffer from hemophilia. All races and races are affected. Hemophilia A is four times more common than hemophilia B, and more than half of people with hemophilia A have severe forms of hemophilia. People with hemophilia need extensive medical monitoring throughout their lives. Without intervention, the affected individual suffers from spontaneous bleeding in the joints, which produces severe pain and debilitating limitation of movement. Bleeding into the muscles causes blood to accumulate in these tissues, and spontaneous bleeding in the throat and neck can cause suffocation if left untreated. Kidney bleeding and postoperative, minor accidental injury or severe bleeding after tooth extraction are also common.

Disclosed herein are formulations for treating bleeding diseases or disorders in a subject in need. The bleeding disorder or condition is selected from the group consisting of bleeding and coagulopathy, hemorrhage in the joints, muscle bleeding, oral bleeding, hemorrhage, bleeding into the muscle, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage , Intrathoracic bleeding, fractures, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal space bleeding, iliopsoas sheath bleeding and any combination thereof. In still other embodiments, the subject is scheduled for surgery. In yet other embodiments, the treatment is prophylactic or on-demand.

Gene therapy using stable and effective lentiviral vector (LV) formulations has been shown to treat patients with hemophilia A by stably integrating factor VIII or factor IX genes into cells, resulting in a sufficient level of functional factor VIII or factor IX. Great prospects for individuals with illness or B.

Somatic cell gene therapy has been explored as a possible treatment for bleeding disorders. Gene therapy has become a particularly attractive treatment method for hemophilia due to its potential to cure the disease by continuously producing FVIII or FIX endogenously after a single administration of the vector encoding the corresponding coagulation factor. Hemophilia A (deficiency of FVIII) and hemophilia B (deficiency of FIX) are very suitable for gene replacement methods, because their clinical manifestations can be completely attributed to the lack of circulating in the plasma in tiny amounts (200 ng/ml). Single gene product (FVIII or FIX).

Lentiviruses have gradually attracted attention as a gene delivery vehicle due to their large capacity and ability to continuously express gene transfer through integration. Lentiviruses have been evaluated in numerous clinical programs for isolated cell therapies, with promising efficacy and safety profiles, and extensive experience gained in the past decade. With the increasing popularity of lentiviruses in in vivo gene therapy, there is a need in the industry to provide improved formulations that enhance the stability of lentiviruses for long-term storage.

The present disclosure satisfies important needs in the industry by providing formulation buffers or vehicles that impart stability to lentiviruses (thus providing long-term frozen storage). In certain exemplary embodiments, where the route of administration is systemic administration, the formulation buffer imparts stability to the lentivirus and provides long-term frozen storage. In some embodiments, the lentivirus is processed into the formulation buffer or vehicle of the present disclosure after purification. After preparation, the lentivirus is stored frozen. The formulation buffers or vehicles of the present invention provide enhanced stability during freezing and thawing and when exposed to elevated temperatures.

Provided herein is a lentiviral vector that contains a codon-optimized FVIII sequence or a codon-optimized FIX sequence, which, when used in gene therapy, exhibits increased performance in a subject and potentially produces Greater therapeutic effect. The embodiment of the present disclosure relates to a lentiviral vector comprising one or more codon-optimized nucleic acid molecules encoding a polypeptide having FVIII activity as described herein, or one or more codon-optimizing nucleic acid molecules comprising one or more codon optimized nucleic acid molecules encoding a polypeptide having FIX activity as described herein The lentiviral vector, the host cell (for example, liver cell) containing the lentiviral vector, and the method of using the disclosed lentiviral vector (for example, the treatment of bleeding disorders using the lentiviral vector disclosed herein). In some embodiments, during the scale-up process, the lentiviral vector is packaged into a lentivirus, and the lentivirus is processed into the formulation buffer or vehicle of the present disclosure.

Generally, the treatment methods disclosed herein involve administration of a lentiviral vector containing a nucleic acid molecule containing at least one codon-optimized nucleic acid sequence encoding FVIII coagulation factor or a nucleic acid molecule containing a nucleic acid molecule containing at least one codon-optimized nucleic acid sequence encoding FIX coagulation factor Lentiviral vector. In some embodiments, the nucleic acid sequence encoding the FVIII coagulation factor is operably linked to a suitable performance control sequence. In some embodiments, the performance control sequence is incorporated into a lentiviral vector (such as a replication-deficient lentiviral vector). )middle. In some embodiments, the nucleic acid sequence encoding the FIX coagulation factor is operably linked to a suitable performance control sequence. In some embodiments, the performance control sequence is incorporated into a lentiviral vector (such as a replication-deficient lentiviral vector). )middle.

The present disclosure provides a method of treating a bleeding disorder (for example, hemophilia A or hemophilia B) in a subject in need, the method comprising administering to the subject at least one dose of lentivirus A vector, the lentiviral vector comprising a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide having FVIII or FIX activity. In certain embodiments, the lentiviral vector is packaged into a lentivirus, and the lentivirus is processed into the formulation buffer of the present invention. In certain embodiments, a nucleotide sequence encoding a polypeptide having FVIII activity includes a nucleotide sequence having a nucleic acid sequence of at least 80%, at least 81%, or at least 82% of the nucleic acid sequence shown in SEQ ID NO: 1 (as shown in Table 1). , At least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88% or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least A nucleotide sequence of 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity. In certain embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity consists of the nucleotide sequence shown in SEQ ID NO: 1 (shown in Table 1). In certain embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity includes a nucleotide sequence having a nucleic acid sequence shown in SEQ ID NO: 2 (shown in Table 1) at least 80%, at least 81%, at least 82%, At least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity nucleotide sequence. In some embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity consists of the nucleotide sequence shown in SEQ ID NO: 2 (shown in Table 1). In certain embodiments, the nucleotide sequence encoding a polypeptide having FIX activity includes a nucleotide sequence with a nucleic acid sequence shown in SEQ ID NO: 3 (shown in Table 1) at least 80%, at least 81%, at least 82%, At least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity nucleotide sequence. In some embodiments, the nucleotide sequence encoding the polypeptide having FIX activity consists of the nucleotide sequence shown in SEQ ID NO: 3 (shown in Table 1).

The present disclosure provides a method of treating a bleeding disorder (for example, hemophilia A or hemophilia B) in a subject in need, the method comprising administering to the subject at least one dose of lentivirus A vector, the lentiviral vector comprising a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide having FVIII or FIX activity. In certain embodiments, the lentiviral vector is packaged into a lentivirus, which is processed into the formulation buffer or vehicle of the invention. In certain embodiments, 5x10 ¹⁰ or less transduction units/kg (TU/kg) (or 10 ⁹ TU/kg or less, or 10 ⁸ TU/kg or less) is administered to the subject At least one dose of the lentiviral vector as described herein, the lentiviral vector comprising a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide having FVIII or FIX activity.

In some embodiments, the dosage is about 5.0x10 ¹⁰ TU/kg, about 4.9x10 ¹⁰ TU/kg, about 4.8x10 ¹⁰ TU/kg, about 4.7x10 ¹⁰ TU/kg, about 4.6x10 ¹⁰ TU/kg, about 4.5 x10 ¹⁰ TU/kg, about 4.4x10 ¹⁰ TU/kg, about 4.3x10 ¹⁰ TU/kg, about 4.2x10 ¹⁰ TU/kg, about 4.1x10 ¹⁰ TU/kg, about 4.0x10 ¹⁰ TU/kg, about 3.9x10 ¹⁰ TU/kg, about 3.8x10 ¹⁰ TU/kg, about 3.7x10 ¹⁰ TU/kg, about 3.6x10 ¹⁰ TU/kg, about 3.5x10 ¹⁰ TU/kg, about 3.4x10 ¹⁰ TU/kg, about 3.3x10 ¹⁰ TU/ kg, from about 3.2x10 ¹⁰ TU / kg, about 3.1x10 ¹⁰ TU / kg, about 3.0x10 ¹⁰ TU / kg, about 2.9x10 ¹⁰ TU / kg, about 2.8x10 ¹⁰ TU / kg, about 2.7x10 ¹⁰ TU / kg, About 2.6x10 ¹⁰ TU/kg, about 2.5x10 ¹⁰ TU/kg, about 2.4x10 ¹⁰ TU/kg, about 2.3x10 ¹⁰ TU/kg, about 2.2x10 ¹⁰ TU/kg, about 2.1x10 ¹⁰ TU/kg, about 2.0 x10 ¹⁰ TU/kg, about 1.9x10 ¹⁰ TU/kg, about 1.8x10 ¹⁰ TU/kg, about 1.7x10 ¹⁰ TU/kg, about 1.6x10 ¹⁰ TU/kg, about 1.5x10 ¹⁰ TU/kg, about 1.4x10 ¹⁰ TU/kg, about 1.3x10 ¹⁰ TU/kg, about 1.2x10 ¹⁰ TU/kg, about 1.1x10 ¹⁰ TU/kg, or about 1.0x10 ¹⁰ TU/kg.

In some embodiments, the dose is about 9.9x10 ⁹ TU / kg, from about 9.8x10 ⁹ TU / kg, from about 9.7x10 ⁹ TU / kg, from about 9.6x10 ⁹ TU / kg, from about 9.5x10 ⁹ TU / kg, from about 9.4 x10 ⁹ TU/kg, about 9.3x10 ⁹ TU/kg, about 9.2x10 ⁹ TU/kg, about 9.1x10 ⁹ TU/kg, about 9.0x10 ⁹ TU/kg, about 8.9x10 ⁹ TU/kg, about 8.8x10 ⁹ TU/kg, about 8.7x10 ⁹ TU/kg, about 8.6x10 ⁹ TU/kg, about 8.5x10 ⁹ TU/kg, about 8.4x10 ⁹ TU/kg, about 8.3x10 ⁹ TU/kg, about 8.2x10 ⁹ TU/ kg, about 8.1x10 ⁹ TU/kg, about 8.0x10 ⁹ TU/kg, about 7.9x10 ⁹ TU/kg, about 7.8x10 ⁹ TU/kg, about 7.7x10 ⁹ TU/kg, about 7.6x10 ⁹ TU/kg, about 7.5x10 ⁹ TU / kg, from about 7.4x10 ⁹ TU / kg, from about 7.3x10 ⁹ TU / kg, from about 7.2x10 ⁹ TU / kg, from about 7.1x10 ⁹ TU / kg, from about 7.0x10 ⁹ TU / kg, from about 6.9 x10 ⁹ TU/kg, about 6.8x10 ⁹ TU/kg, about 6.7x10 ⁹ TU/kg, about 6.6x10 ⁹ TU/kg, about 6.5x10 ⁹ TU/kg, about 6.4x10 ⁹ TU/kg, about 6.3x10 ⁹ TU/kg, about 6.2x10 ⁹ TU/kg, about 6.1x10 ⁹ TU/kg, about 6.0x10 ⁹ TU/kg, about 5.9x10 ⁹ TU/kg, about 5.8x10 ⁹ TU/kg, about 5.7x10 ⁹ TU/ kg, about 5.6x10 ⁹ TU/kg, about 5.5x10 ⁹ TU/kg, about 5.4x10 ⁹ TU/kg, about 5.3x10 ⁹ TU/kg, about 5.2x10 ⁹ TU/kg, about 5.1x10 ⁹ TU/kg, About 5.0x10 ⁹ TU/kg, about 4.9x10 ⁹ TU/kg, about 4.8x10 ⁹ TU/kg, about 4.7x10 ⁹ TU/kg, about 4.6x10 ⁹ TU/kg, about 4.5x10 ⁹ TU/kg, about 4.4 x10 ⁹ TU/kg, about 4.3x10 ⁹ TU/kg, about 4.2x10 ⁹ TU/kg, about 4.1x10 ⁹ TU/kg, about 4.0x10 ⁹ TU/kg, about 3.9x10 ⁹ TU/kg, about 3.8x10 ⁹ TU/ kg, about 3.7x10 ⁹ TU/kg, about 3.6x10 ⁹ TU/kg, about 3.5x10 ⁹ TU/kg, about 3.4x10 ⁹ TU/kg, about 3.3x10 ⁹ TU/kg, about 3.2x10 ⁹ TU/kg, about 3.1x10 ⁹ TU / kg, from about 3.0x10 ⁹ TU / kg, from about 2.9x10 ⁹ TU / kg, from about 2.8x10 ⁹ TU / kg, from about 2.7x10 ⁹ TU / kg, from about 2.6x10 ⁹ TU / kg, from about 2.5 x10 ⁹ TU/kg, about 2.4x10 ⁹ TU/kg, about 2.3x10 ⁹ TU/kg, about 2.2x10 ⁹ TU/kg, about 2.1x10 ⁹ TU/kg, about 2.0x10 ⁹ TU/kg, about 1.9x10 ⁹ TU/kg, about 1.8x10 ⁹ TU/kg, about 1.7x10 ⁹ TU/kg, about 1.6x10 ⁹ TU/kg, about 1.5x10 ⁹ TU/kg, about 1.4x10 ⁹ TU/kg, about 1.3x10 ⁹ TU/ kg, about 1.2x10 ⁹ TU/kg, about 1.1x10 ⁹ TU/kg, or about 1.0x10 ⁹ TU/kg.

In some embodiments, the dose is about 9.9x10 ⁸ TU / kg, from about 9.8x10 ⁸ TU / kg, from about 9.7x10 ⁸ TU / kg, from about 9.6x10 ⁸ TU / kg, from about 9.5x10 ⁸ TU / kg, from about 9.4 x10 ⁸ TU/kg, about 9.3x10 ⁸ TU/kg, about 9.2x10 ⁸ TU/kg, about 9.1x10 ⁸ TU/kg, about 9.0x10 ⁸ TU/kg, about 8.9x10 ⁸ TU/kg, about 8.8x10 ⁸ TU/kg, about 8.7x10 ⁸ TU/kg, about 8.6x10 ⁸ TU/kg, about 8.5x10 ⁸ TU/kg, about 8.4x10 ⁸ TU/kg, about 8.3x10 ⁸ TU/kg, about 8.2x10 ⁸ TU/ kg, about 8.1x10 ⁸ TU/kg, about 8.0x10 ⁸ TU/kg, about 7.9x10 ⁸ TU/kg, about 7.8x10 ⁸ TU/kg, about 7.7x10 ⁸ TU/kg, about 7.6x10 ⁸ TU/kg, about 7.5x10 ⁸ TU / kg, from about 7.4x10 ⁸ TU / kg, from about 7.3x10 ⁸ TU / kg, from about 7.2x10 ⁸ TU / kg, from about 7.1x10 ⁸ TU / kg, from about 7.0x10 ⁸ TU / kg, from about 6.9 x10 ⁸ TU/kg, about 6.8x10 ⁸ TU/kg, about 6.7x10 ⁸ TU/kg, about 6.6x10 ⁸ TU/kg, about 6.5x10 ⁸ TU/kg, about 6.4x10 ⁸ TU/kg, about 6.3x10 ⁸ TU/kg, about 6.2x10 ⁸ TU/kg, about 6.1x10 ⁸ TU/kg, about 6.0x10 ⁸ TU/kg, about 5.9x10 ⁸ TU/kg, about 5.8x10 ⁸ TU/kg, about 5.7x10 ⁸ TU/ kg, about 5.6x10 ⁸ TU/kg, about 5.5x10 ⁸ TU/kg, about 5.4x10 ⁸ TU/kg, about 5.3x10 ⁸ TU/kg, about 5.2x10 ⁸ TU/kg, about 5.1x10 ⁸ TU/kg, About 5.0x10 ⁸ TU/kg, about 4.9x10 ⁸ TU/kg, about 4.8x10 ⁸ TU/kg, about 4.7x10 ⁸ TU/kg, about 4.6x10 ⁸ TU/kg, about 4.5x10 ⁸ TU/kg, about 4.4 x10 ⁸ TU/kg, about 4.3x10 ⁸ TU/kg, about 4.2x10 ⁸ TU/kg, about 4.1x10 ⁸ TU/kg, about 4.0x10 ⁸ TU/kg, about 3.9x10 ⁸ TU/kg, about 3.8x10 ⁸ TU/ kg, about 3.7x10 ⁸ TU/kg, about 3.6x10 ⁸ TU/kg, about 3.5x10 ⁸ TU/kg, about 3.4x10 ⁸ TU/kg, about 3.3x10 ⁸ TU/kg, about 3.2x10 ⁸ TU/kg, about 3.1x10 ⁸ TU / kg, from about 3.0x10 ⁸ TU / kg, from about 2.9x10 ⁸ TU / kg, from about 2.8x10 ⁸ TU / kg, from about 2.7x10 ⁸ TU / kg, from about 2.6x10 ⁸ TU / kg, from about 2.5 x10 ⁸ TU/kg, about 2.4x10 ⁸ TU/kg, about 2.3x10 ⁸ TU/kg, about 2.2x10 ⁸ TU/kg, about 2.1x10 ⁸ TU/kg, about 2.0x10 ⁸ TU/kg, about 1.9x10 ⁸ TU/kg, about 1.8x10 ⁸ TU/kg, about 1.7x10 ⁸ TU/kg, about 1.6x10 ⁸ TU/kg, about 1.5x10 ⁸ TU/kg, about 1.4x10 ⁸ TU/kg, about 1.3x10 ⁸ TU/ kg, about 1.2x10 ⁸ TU/kg, about 1.1x10 ⁸ TU/kg, or about 1.0x10 ⁸ TU/kg.

In some embodiments, the dose is less than 5.0x10 ¹⁰ TU / kg, less than 4.9x10 ¹⁰ TU / kg, less than 4.8x10 ¹⁰ TU / kg, less than 4.7x10 ¹⁰ TU / kg, less than 4.6x10 ¹⁰ TU / kg, less than 4.5x10 ¹⁰ TU/kg, less than 4.4x10 ¹⁰ TU/kg, less than 4.3x10 ¹⁰ TU/kg, less than 4.2x10 ¹⁰ TU/kg, less than 4.1x10 ¹⁰ TU/kg, less than 4.0x10 ¹⁰ TU/kg, less than 3.9x10 ¹⁰ TU /kg, less than 3.8x10 ¹⁰ TU/kg, less than 3.7x10 ¹⁰ TU/kg, less than 3.6x10 ¹⁰ TU/kg, less than 3.5x10 ¹⁰ TU/kg, less than 3.4x10 ¹⁰ TU/kg, less than 3.3x10 ¹⁰ TU/kg , less than 3.2x10 ¹⁰ TU / kg, less than 3.1x10 ¹⁰ TU / kg, less than 3.0x10 ¹⁰ TU / kg, less than 2.9x10 ¹⁰ TU / kg, less than 2.8x10 ¹⁰ TU / kg, less than 2.7x10 ¹⁰ TU / kg, less than 2.6x10 ¹⁰ TU / kg, less than 2.5x10 ¹⁰ TU / kg, less than 2.4x10 ¹⁰ TU / kg, less than 2.3x10 ¹⁰ TU / kg, less than 2.2x10 ¹⁰ TU / kg, less than 2.1x10 ¹⁰ TU / kg, less than 2.0x10 ¹⁰ TU / kg, less than 1.9x10 ¹⁰ TU / kg, less than 1.8x10 ¹⁰ TU / kg, less than 1.7x10 ¹⁰ TU / kg, less than 1.6x10 ¹⁰ TU / kg, less than 1.5x10 ¹⁰ TU / kg, less than 1.4x10 ¹⁰ TU /kg, less than 1.3x10 ¹⁰ TU/kg, less than 1.2x10 ¹⁰ TU/kg, less than 1.1x10 ¹⁰ TU/kg, or less than 1.0x10 ¹⁰ TU/kg.

In some embodiments, the dose is less than 9.9x10 ⁹ TU / kg, less than 9.8x10 ⁹ TU / kg, less than 9.7x10 ⁹ TU / kg, less than 9.6x10 ⁹ TU / kg, less than 9.5x10 ⁹ TU / kg, less than 9.4x10 ⁹ TU/kg, less than 9.3x10 ⁹ TU/kg, less than 9.2x10 ⁹ TU/kg, less than 9.1x10 ⁹ TU/kg, less than 9.0x10 ⁹ TU/kg, less than 8.9x10 ⁹ TU/kg, less than 8.8x10 ⁹ TU /kg, less than 8.7x10 ⁹ TU/kg, less than 8.6x10 ⁹ TU/kg, less than 8.5x10 ⁹ TU/kg, less than 8.4x10 ⁹ TU/kg, less than 8.3x10 ⁹ TU/kg, less than 8.2x10 ⁹ TU/kg , Less than 8.1x10 ⁹ TU/kg, less than 8.0x10 ⁹ TU/kg, less than 7.9x10 ⁹ TU/kg, less than 7.8x10 ⁹ TU/kg, less than 7.7x10 ⁹ TU/kg, less than 7.6x10 ⁹ TU/kg, less than 7.5x10 ⁹ TU / kg, less than 7.4x10 ⁹ TU / kg, less than 7.3x10 ⁹ TU / kg, less than 7.2x10 ⁹ TU / kg, less than 7.1x10 ⁹ TU / kg, less than 7.0x10 ⁹ TU / kg, less than 6.9x10 ⁹ TU/kg, less than 6.8x10 ⁹ TU/kg, less than 6.7x10 ⁹ TU/kg, less than 6.6x10 ⁹ TU/kg, less than 6.5x10 ⁹ TU/kg, less than 6.4x10 ⁹ TU/kg, less than 6.3x10 ⁹ TU / kg, less than 6.2x10 ⁹ TU / kg, less than 6.1x10 ⁹ TU / kg, less than 6.0x10 ⁹ TU / kg, less than 5.9x10 ⁹ TU / kg, less than 5.8x10 ⁹ TU / kg, less than 5.7x10 ⁹ TU / kg , Less than 5.6x10 ⁹ TU/kg, less than 5.5x10 ⁹ TU/kg, less than 5.4x10 ⁹ TU/kg, less than 5.3x10 ⁹ TU/kg, less than 5.2x10 ⁹ TU/kg, less than 5.1x10 ⁹ TU/kg, less than 5.0x10 ⁹ TU / kg, less than 4.9x10 ⁹ TU / kg, less than 4.8x10 ⁹ TU / kg, less than 4.7x10 ⁹ TU / kg, less than 4.6x10 ⁹ TU / kg, less than 4.5x10 ⁹ TU / kg, less than 4.4x10 ⁹ TU/kg, less than 4.3x10 ⁹ TU/kg, less than 4.2x10 ⁹ TU/kg, less than 4. 1x10 ⁹ TU/kg, less than 4.0x10 ⁹ TU/kg, less than 3.9x10 ⁹ TU/kg, less than 3.8x10 ⁹ TU/kg, less than 3.7x10 ⁹ TU/kg, less than 3.6x10 ⁹ TU/kg, less than 3.5x10 ⁹ TU / kg, less than 3.4x10 ⁹ TU / kg, less than 3.3x10 ⁹ TU / kg, less than 3.2x10 ⁹ TU / kg, less than 3.1x10 ⁹ TU / kg, less than 3.0x10 ⁹ TU / kg, less than 2.9x10 ⁹ TU / kg, less than 2.8x10 ⁹ TU/kg, less than 2.7x10 ⁹ TU/kg, less than 2.6x10 ⁹ TU/kg, less than 2.5x10 ⁹ TU/kg, less than 2.4x10 ⁹ TU/kg, less than 2.3x10 ⁹ TU/kg, less than 2.2x10 ⁹ TU / kg, less than 2.1x10 ⁹ TU / kg, less than 2.0x10 ⁹ TU / kg, less than 1.9x10 ⁹ TU / kg, less than 1.8x10 ⁹ TU / kg, less than 1.7x10 ⁹ TU / kg, less than 1.6 x10 ⁹ TU / kg, less than 1.5x10 ⁹ TU / kg, less than 1.4x10 ⁹ TU / kg, less than 1.3x10 ⁹ TU / kg, less than 1.2x10 ⁹ TU / kg, less than 1.1x10 ⁹ TU / kg, or less than 1.0x10 ⁹ TU/kg.

In some embodiments, the dose is less than 9.9x10 ⁸ TU / kg, less than 9.8x10 ⁸ TU / kg, less than 9.7x10 ⁸ TU / kg, less than 9.6x10 ⁸ TU / kg, less than 9.5x10 ⁸ TU / kg, less than 9.4x10 ⁸ TU/kg, less than 9.3x10 ⁸ TU/kg, less than 9.2x10 ⁸ TU/kg, less than 9.1x10 ⁸ TU/kg, less than 9.0x10 ⁸ TU/kg, less than 8.9x10 ⁸ TU/kg, less than 8.8x10 ⁸ TU /kg, less than 8.7x10 ⁸ TU/kg, less than 8.6x10 ⁸ TU/kg, less than 8.5x10 ⁸ TU/kg, less than 8.4x10 ⁸ TU/kg, less than 8.3x10 ⁸ TU/kg, less than 8.2x10 ⁸ TU/kg , Less than 8.1x10 ⁸ TU/kg, less than 8.0x10 ⁸ TU/kg, less than 7.9x10 ⁸ TU/kg, less than 7.8x10 ⁸ TU/kg, less than 7.7x10 ⁸ TU/kg, less than 7.6x10 ⁸ TU/kg, less than 7.5x10 ⁸ TU / kg, less than 7.4x10 ⁸ TU / kg, less than 7.3x10 ⁸ TU / kg, less than 7.2x10 ⁸ TU / kg, less than 7.1x10 ⁸ TU / kg, less than 7.0x10 ⁸ TU / kg, less than 6.9x10 ⁸ TU/kg, less than 6.8x10 ⁸ TU/kg, less than 6.7x10 ⁸ TU/kg, less than 6.6x10 ⁸ TU/kg, less than 6.5x10 ⁸ TU/kg, less than 6.4x10 ⁸ TU/kg, less than 6.3x10 ⁸ TU / kg, less than 6.2x10 ⁸ TU / kg, less than 6.1x10 ⁸ TU / kg, less than 6.0x10 ⁸ TU / kg, less than 5.9x10 ⁸ TU / kg, less than 5.8x10 ⁸ TU / kg, less than 5.7x10 ⁸ TU / kg , Less than 5.6x10 ⁸ TU/kg, less than 5.5x10 ⁸ TU/kg, less than 5.4x10 ⁸ TU/kg, less than 5.3x10 ⁸ TU/kg, less than 5.2x10 ⁸ TU/kg, less than 5.1x10 ⁸ TU/kg, less than 5.0x10 ⁸ TU / kg, less than 4.9x10 ⁸ TU / kg, less than 4.8x10 ⁸ TU / kg, less than 4.7x10 ⁸ TU / kg, less than 4.6x10 ⁸ TU / kg, less than 4.5x10 ⁸ TU / kg, less than 4.4x10 ⁸ TU/kg, less than 4.3x10 ⁸ TU/kg, less than 4.2x10 ⁸ TU/kg, less than 4. 1x10 ⁸ TU/kg, less than 4.0x10 ⁸ TU/kg, less than 3.9x10 ⁸ TU/kg, less than 3.8x10 ⁸ TU/kg, less than 3.7x10 ⁸ TU/kg, less than 3.6x10 ⁸ TU/kg, less than 3.5x10 ⁸ TU / kg, less than 3.4x10 ⁸ TU / kg, less than 3.3x10 ⁸ TU / kg, less than 3.2x10 ⁸ TU / kg, less than 3.1x10 ⁸ TU / kg, less than 3.0x10 ⁸ TU / kg, less than 2.9x10 ⁸ TU / kg, less than 2.8x10 ⁸ TU/kg, less than 2.7x10 ⁸ TU/kg, less than 2.6x10 ⁸ TU/kg, less than 2.5x10 ⁸ TU/kg, less than 2.4x10 ⁸ TU/kg, less than 2.3x10 ⁸ TU/kg, less than 2.2x10 ⁸ TU / kg, less than 2.1x10 ⁸ TU / kg, less than 2.0x10 ⁸ TU / kg, less than 1.9x10 ⁸ TU / kg, less than 1.8x10 ⁸ TU / kg, less than 1.7x10 ⁸ TU / kg, less than 1.6 x10 ⁸ TU / kg, less than 1.5x10 ⁸ TU / kg, less than 1.4x10 ⁸ TU / kg, less than 1.3x10 ⁸ TU / kg, less than 1.2x10 ⁸ TU / kg, less than 1.1x10 ⁸ TU / kg, or less than 1.0x10 ⁸ TU/kg.

In some embodiments, the dose is between 1 ^{×10 8} TU/kg and 5× ^{10 10} TU/kg, between 1.5 ^{×10 8} TU/kg and 5× ^{10 10} TU/kg, between 2 ^{×10 8} TU/kg and 5× ^{10 10} TU/kg Between 2.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 3x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 3.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, Between 4x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 4.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 5.5x10 ⁸ Between TU/kg and 5x10 ¹⁰ TU/kg, between 6x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 6.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 7x10 ⁸ TU/kg and Between 5x10 ¹⁰ TU/kg, between 7.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 8x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 8.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU /kg, between 9x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 9.5x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 1x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg , Between 1.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 2x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 2.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 3x10 ^{Between 9} TU/kg and 5x10 ¹⁰ TU/kg, between 3.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 4x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 4.5x10 ⁹ TU/kg kg and 5x10 ¹⁰ TU/kg, between 5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 5.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 6x10 ⁹ TU/kg and 5x10 ¹⁰ Between TU/kg, between 6.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 7x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 7.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg Of Between 8x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 8.5x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 9x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 9.5 x10 ⁹ TU/kg and 5x10 ¹⁰ TU/kg, between 10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg, between 1.5x10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg, between 2x10 ¹⁰ TU/kg kg and 5x10 ¹⁰ TU/kg, between 2.5x10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg, between 3x10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg, between 3.5x10 ¹⁰ TU/kg and 5x10 ^{Between 10} TU/kg, between 4x10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg, or between 4.5x10 ¹⁰ TU/kg and 5x10 ¹⁰ TU/kg.

In some embodiments, the dose is between 1x10 ⁸ TU/kg and 5x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 4.5x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 4x10 ¹⁰ TU/kg Between 1x10 ⁸ TU/kg and 3.5x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 3x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 2.5x10 ¹⁰ TU/kg, Between 1x10 ⁸ TU/kg and 2x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 1.5x10 ¹⁰ TU/kg, between 1x10 ⁸ TU/kg and 10 ¹⁰ TU/kg, between 1x10 ⁸ TU /kg and 9x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 8.5x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 8x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 7.5 between x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 7x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 6.5x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 6x10 ⁹ TU/ kg, between 1x10 ⁸ TU/kg and 5.5x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 5x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 4.5x10 ⁹ TU/kg , Between 1x10 ⁸ TU/kg and 4x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 3.5x10 ⁹ TU/kg, between 1x10 ⁸ TU/kg and 3x10 ⁹ TU/kg, between 1x10 ⁸ between TU / kg and 2.5x10 ⁹ TU / kg, at 1x10 ⁸ TU / kg between 2x10 ⁹ and between 1x10 ⁸ TU / kg and 1.5x10 ⁹ TU / kg, at 1x10 ⁸ TU / kg and 1x10 ⁹ Between TU/kg, between 1x10 ⁸ TU/kg and 9.5x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 9x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 8.5x10 ⁸ TU/kg Between 1x10 ⁸ TU/kg and 8x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 7.5x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 7x10 ⁸ TU/kg, between Between 1x10 ⁸ TU/kg and 6.5x10 ⁸ TU/kg, Between 1x10 ⁸ TU/kg and 6x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 5.5x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 5x10 ⁸ TU/kg, between 1x10 ⁸ TU /kg and 4.5x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 4x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and 3.5x10 ⁸ TU/kg, between 1x10 ⁸ TU/kg and between 3x10 ⁸ TU / kg, between 1x10 ⁸ TU / kg and 2.5x10 ⁸ TU / kg, at 1x10 ⁸ TU / kg between 2x10 ^8, or 1x10 ⁸ TU / kg and 1.5x10 ⁸ TU / kg between.

In some embodiments, the dose is between 1x10 ¹⁰ TU/kg and 2x10 ¹⁰ TU/kg, between 1.1x10 ¹⁰ TU/kg and 1.9x10 ¹⁰ TU/kg, between 1.2x10 ¹⁰ TU/kg and 1.8x10 ¹⁰ between TU / kg, between 1.3x10 ¹⁰ TU / kg and 1.7x10 ¹⁰ TU / kg, or between 1.4x10 ¹⁰ TU / kg and 1.6x10 ¹⁰ TU / kg. In some embodiments, the dosage is about 1.5x10 ¹⁰ TU/kg. In some embodiments, the dosage is 1.5x10 ¹⁰ TU/kg.

In some embodiments, the dose is between 1x10 ⁹ TU / kg and 2x10 ⁹ TU / kg, between 1.1x10 ⁹ TU / kg and 1.9x10 ⁹ TU / kg, at 1.2x10 ⁹ TU / kg and 1.8x10 ⁹ between TU / kg, between 1.3x10 ⁹ TU / kg and 1.7x10 ⁹ TU / kg, or between 1.4x10 ⁹ TU / kg and 1.6x10 ⁹ TU / kg. In some embodiments, the dosage is 1.5x10 ⁹ TU/kg. In certain embodiments, the dosage is about 3.0 x 10 ⁹ TU/kg.

In some embodiments, the plasma FVIII activity increases 24 hours, 36 hours, or 48 hours after the administration of the lentiviral vector of the present disclosure relative to the plasma FVIII activity in the subject administered the control lentiviral vector. In some embodiments, relative to the plasma FVIII activity in the subject administered the control nucleic acid molecule, the plasma FVIII activity increases 24 hours, 36 hours, or 48 hours after administration of the lentiviral vector of the present disclosure.

In some embodiments, the plasma FIX activity increases 24 hours, 36 hours, or 48 hours after the administration of the lentiviral vector of the present disclosure relative to the plasma FIX activity in the subject administered the control lentiviral vector. In some embodiments, the plasma FIX activity increases 24 hours, 36 hours, or 48 hours after the administration of the lentiviral vector of the present disclosure relative to the plasma FIX activity in the subject administered the control nucleic acid molecule.

In some embodiments, relative to a subject administered a control lentiviral vector or a control nucleic acid molecule, about 6 hours, about 12 hours, about 18 hours, about 24 hours after the administration of the lentiviral vector of the present disclosure , About 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days , About 26 days, about 27 days, or about 28 days, plasma FVIII or plasma FIX activity increases.

In some embodiments, regarding the basal level in the subject, the plasma FVIII or plasma FIX activity in the subject is increased by at least About 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least About 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least About 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least About 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or At least about 200 times.

In some embodiments, the lentiviral vector is administered in a single dose or multiple doses. In some embodiments, the dose of lentiviral vector is administered at one time or divided into multiple sub-doses, such as two sub-doses, three sub-doses, four sub-doses, five sub-doses, six sub-doses, or more than six sub-doses. Sub-dose. In some embodiments, more than one lentiviral vector is administered.

In some embodiments, the dose of the lentiviral vector is repeatedly administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times . In some embodiments, the lentiviral vector is administered via intravenous injection.

In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is an adult subject.

In some embodiments, the lentiviral vector contains at least one tissue-specific promoter, that is, a promoter that regulates the expression of a polypeptide having FVIII activity or a polypeptide having FIX activity in a specific tissue or cell type. In some embodiments, the tissue-specific promoter in the lentiviral vector selectively enhances the expression of the polypeptide having FVIII activity in target hepatocytes. In some embodiments, the tissue-specific promoter that selectively enhances the expression of a polypeptide having FVIII activity in target hepatocytes includes the mTTR promoter. In some embodiments, tissue-specific promoters that selectively enhance the expression of polypeptides with FIX activity in target hepatocytes include APOA2 promoter, SERPINA1 (hAAT) promoter, mTTR promoter, MIR122 promoter, ET promoter (GenBank number AY661265; see also Vigna et al., Molecular Therapy 11(5) :763 (2005)) or any combination thereof. In some embodiments, the target hepatocytes are hepatocytes.

Since lentiviral vectors can transduce all liver cell types, different promoters can be used in lentiviral vectors to control the performance of gene transfer (for example, FVIII or FIX) in different cell types. Therefore, the lentiviral vector may contain a specific promoter that will control the performance of the FVIII gene transfer or the FIX gene transfer in different tissues or cell types (such as different liver tissues or cell types). Therefore, in some embodiments, the lentiviral vector may include an endothelial-specific promoter that will control the expression of FVIII gene transfer or FIX gene transfer in liver endothelial tissue, or will control FVIII gene transfer or FIX gene transfer. Hepatocyte-specific promoters expressed in hepatocytes, or both.

In some embodiments, the lentiviral vector contains one or more tissue-specific promoters that control the expression of FVIII gene transfer or FIX gene transfer in tissues other than the liver. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of the target cell or target tissue, for example, in the genome of liver cells or in the genome of liver endothelial cells.

In some embodiments, the isolated nucleic acid molecule in the lentiviral vector of the present disclosure further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence (eg, a half-life extender). In some embodiments, the heterologous amino acid sequence is an immunoglobulin constant region or portion thereof, XTEN, transferrin, albumin, or PAS sequence. In some embodiments, the heterologous amino acid sequence is connected to the N-terminus or C-terminus of the amino acid sequence encoded by the nucleotide sequence, or the nucleoside is inserted at one or more insertion sites selected from Table 2. Between two amino acids in the amino acid sequence encoded by the acid sequence. The heterologous nucleotide sequence is described further herein.

In some embodiments, the polypeptide having FVIII activity is human FVIII. In some embodiments, the polypeptide having FVIII activity is full-length FVIII. In some embodiments, the polypeptide having FVIII activity is FVIII with B domain deleted.

In some embodiments, the polypeptide having FIX activity is human FIX. In some embodiments, the polypeptide having FIX activity is full-length FIX. In some embodiments, the polypeptide having FIX activity is a variant of human FIX. In certain embodiments, the polypeptide having FIX activity is an R338L variant of human FIX. In certain embodiments, the polypeptide having FIX activity is a Padua variant.

The lentiviral vector disclosed herein can be used in mammals (such as human patients). It will be therapeutically beneficial to use gene therapy to treat bleeding diseases or disorders selected from the following: hemorrhage and coagulopathy, hemorrhage in joints, hemorrhage in muscles, Oral bleeding, hemorrhage, bleeding into the muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system hemorrhage, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space and Bleeding of the iliopsoas sheath. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. In another embodiment, the bleeding disease or disorder is hemophilia B.

In some embodiments, prior to administration to a patient, target cells (eg, liver cells) are treated in vitro with the lentiviral vectors disclosed herein. In certain embodiments, prior to administration to a patient, target cells (eg, liver cells) are treated in vitro with the lentiviral vectors disclosed herein. In yet another embodiment, prior to administration to the patient, cells from the patient (eg, hepatocytes) are treated ex vivo with the lentiviral vector disclosed herein.

In some embodiments, relative to the physiologically normal circulating FVIII levels, the lentiviral vectors disclosed herein are administered (e.g., at 10 ¹⁰ TU/kg or lower, 10 ⁹ TU/kg or lower, or 10 ⁸ TU /kg or lower administration), plasma FVIII activity increased by at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, At least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, At least about 270%, at least about 280%, at least about 290%, or at least about 300%.

In some embodiments, relative to the physiologically normal circulating FIX levels, the lentiviral vectors disclosed herein are administered (e.g., at 10 ¹⁰ TU/kg or lower, 10 ⁹ TU/kg or lower, or 10 ⁸ TU /kg or lower administration), the plasma FIX activity increased by at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, At least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, At least about 270%, at least about 280%, at least about 290%, or at least about 300%.

In one embodiment, the plasma FVIII activity is increased by at least about 3,000% to about 5,000% after administration of the lentiviral vector of the present disclosure relative to the physiologically normal circulating FVIII level. In some embodiments, relative to a subject administered a control lentiviral vector or a control nucleic acid molecule, the lentivirus described herein comprising a codon-optimized gene encoding a polypeptide having factor VIII (FVIII) activity is administered After carrier, plasma FVIII activity is increased by at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times.

In one embodiment, the plasma FIX activity is increased by at least about 3,000% to about 5,000% after administration of the lentiviral vector of the present disclosure relative to physiologically normal circulating FIX levels. In some embodiments, relative to a subject administered a control lentiviral vector or a control nucleic acid molecule, the lentivirus described herein comprising a codon-optimized gene encoding a polypeptide having factor IX (FIX) activity is administered After carrier, the plasma FIX activity is increased by at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times.

The present disclosure also provides a method for treating, preventing or ameliorating a hemostatic disorder (for example, a bleeding disorder such as hemophilia A or hemophilia B) in a subject in need, the method comprising: The subject is administered a therapeutically effective amount of a lentiviral vector, the lentiviral vector comprising an isolated nucleic acid molecule containing a nucleotide sequence encoding a polypeptide having FVIII activity or a polypeptide having FIX activity.

The treatment, improvement and prevention by the lentiviral vector of the present disclosure can be bypass therapy. Subjects undergoing bypass therapy may have developed inhibitors against coagulation factors (for example, FVIII or FIX), or are developing coagulation factor inhibitors.

The lentiviral vector of the present disclosure treats or prevents blood disorders by promoting the formation of fibrin clots. The polypeptides with FVIII or FIX activity encoded by the nucleic acid molecules of the present disclosure can initiate members of the coagulation cascade. The coagulation factor can be a participant in the external pathway, the internal pathway, or both.

The lentiviral vectors of the present disclosure can be used to treat hemostatic disorders that are known to be treatable with FVIII or FIX. Hemostatic disorders that can be treated using the methods of the present disclosure include, but are not limited to, hemophilia A, hemophilia B, von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, and fibrinogen, Absence or structural abnormality of prothrombin, factor V, factor VII, factor X or factor XIII, hemorrhage in the joints, muscle bleeding, oral bleeding, hemorrhage, bleeding into the muscle, oral hemorrhage, trauma, head trauma, gastrointestinal Bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system hemorrhage, retropharyngeal space hemorrhage, retroperitoneal space hemorrhage and iliopsoas muscle sheath hemorrhage.

The composition for administration to a subject includes a lentiviral vector comprising a nucleic acid molecule containing the optimized nucleotide sequence of the present disclosure, the optimized nucleotide sequence encoding FVIII coagulation factor or FIX coagulation Factors (for gene therapy applications) and FVIII or FIX polypeptide molecules. In some embodiments, the composition for administration is a cell contacted with the lentiviral vector of the present disclosure in vivo, in vitro, or ex vivo.

In some embodiments, the hemostatic disorder is a genetic disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is the result of a lack of FVIII. In other embodiments, the hemostatic disorder may be the result of a defective FVIII clotting factor. In one embodiment, the subject has hemophilia B. In other embodiments, the hemostatic disorder is the result of a lack of FIX. In other embodiments, the hemostatic disorder may be the result of a defective FIX clotting factor.

In another embodiment, the hemostatic disorder may be an acquired disorder. Acquired disorders may originate from underlying secondary diseases or conditions. The unrelated condition can be (by way of example but not limitation) cancer, autoimmune disease or pregnancy. Acquired disorders may result from aging or from drug therapies to treat potential secondary disorders (eg, cancer chemotherapy).

The present disclosure also relates to methods of treating subjects who do not suffer from a hemostatic disorder or a secondary disease or condition that leads to the hemostatic disorder. The present disclosure therefore relates to a method of treating a subject in need of a universal hemostatic agent, the method comprising administering a therapeutically effective amount of the lentiviral vector of the present disclosure. For example, in one embodiment, a subject in need of a universal hemostatic agent is undergoing or is about to undergo surgery. The lentiviral vector of the present disclosure can be administered as a preventive agent before or after surgery.

The lentiviral vectors of the present disclosure can be administered during or after surgery to control acute bleeding episodes. Surgery may include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation.

In another embodiment, the lentiviral vector of the present disclosure can be used to treat subjects with acute bleeding episodes who do not suffer from hemostatic disorders. Acute bleeding episodes may result from severe trauma (for example, surgery), car accidents, wounds, laceration gun shots, or any other traumatic event that causes uncontrolled bleeding.

Lentiviral vectors can be used to preventively treat subjects suffering from hemostatic disorders. Lentiviral vectors can also be used to treat acute bleeding episodes in subjects suffering from hemostatic disorders.

In another embodiment, the administration of the lentiviral vector disclosed herein and/or the subsequent manifestation of the FVIII protein or FIX protein does not induce an immune response in the subject. In some embodiments, the immune response comprises the production of antibodies against FVIII or FIX. In some embodiments, the immune response includes cytokine secretion. In some embodiments, the immune response comprises priming B cells, T cells, or both B cells and T cells. In some embodiments, the immune response is an inhibitory immune response, wherein the immune response of the subject reduces the activity of the FVIII protein relative to the activity of FVIII in a subject that has not developed an immune response. In certain embodiments, expressing FVIII protein by administering a lentiviral vector of the present disclosure prevents an inhibitory immune response against FVIII protein or FVIII protein expressed from an isolated nucleic acid molecule or a lentiviral vector. In some embodiments, the immune response is an inhibitory immune response, wherein the immune response of the subject reduces the activity of the FIX protein relative to the activity of FIX in a subject that has not developed an immune response. In certain embodiments, the expression of FIX protein by administering the lentiviral vector of the present disclosure prevents the inhibitory immune response against the FIX protein or the FIX protein expressed from the isolated nucleic acid molecule or the lentiviral vector.

In some embodiments, the lentiviral vector of the present disclosure is administered in combination with at least one other agent that promotes hemostasis. The other agents that promote hemostasis are therapeutic agents that have proven coagulation activity. By way of example but not limitation, the hemostatic agent may include factor V, factor VII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin or fibrinogen or any of the foregoing initiation forms. The coagulation factor or hemostatic agent may also include anti-fibrinolytic drugs, such as epsilon-aminocaproic acid, tranexamic acid.

In one embodiment of the present disclosure, the composition (eg, a lentiviral vector) is a composition in which FVIII exists in a form that can be activated when administered to a subject. In one embodiment of the present disclosure, the composition (for example, a lentiviral vector) is a composition in which FIX exists in a form that can be activated when administered to a subject. This activatable molecule can be activated at the coagulation site in the body after being administered to the subject.

The lentiviral vector of the present disclosure can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, such as oral , sublingual, transbuccal, sublingual, transnasal, transrectal, transvaginal or transpulmonary routes Vote. The lentiviral vector can be implanted into or connected to the solid-phase support of the biopolymer, thereby allowing the slow release of the vector to the desired site.

In one embodiment, the route of administration of the lentiviral vector is parenteral. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous form of parenteral administration is preferred. Although all these administration forms are explicitly considered within the scope of the present disclosure, the administration form may be a solution for injection, especially for intravenous or intraarterial injection or drip infusion. Generally, pharmaceutical compositions suitable for injection may contain buffers (such as acetate, phosphate or citrate buffers), surfactants (such as polysorbates), and optionally stabilizers (such as human albumin). Wait. However, in other methods compatible with the teachings herein, the lentiviral vector can be delivered directly to the site of the undesirable cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the present disclosure, pharmaceutically acceptable carriers include but are not limited to 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

More specifically, pharmaceutical compositions suitable for injection use include sterile aqueous solutions (in the case of water solubility) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy syringability is possible. It should be stable under manufacturing and storage conditions, and will better resist the contaminating action of microorganisms (such as bacteria and fungi) and preserve it. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants.

The effect of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption (for example, aluminum monostearate and gelatin).

In any case, a sterile injectable solution can be prepared by incorporating the active compound (for example, the polypeptide itself or in combination with other active agents) in the required amount into a product having one or a combination of the ingredients listed above as necessary. In a suitable solvent, filter and sterilize afterwards. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from the previously sterile filtered solution . The preparations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the formulations can be packaged and sold in kits. Such products preferably have a label or package insert indicating that the relevant composition can be used to treat subjects suffering from or susceptible to coagulopathy.

The pharmaceutical composition may also be formulated for rectal administration as, for example, a suppository containing a conventional suppository base (such as cocoa butter or other glycerides) or a retention enemas.

The effective dose of the composition of the present disclosure for the treatment of disorders varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, and other drugs administered And whether the treatment is preventive or curative. Generally, the patient is a human, but non-human mammals can also be treated, including transgenic mammals. The therapeutic dose can be gradually increased using conventional methods known to those skilled in the art to optimize safety and efficacy.

The lentiviral vector can be administered in a single dose or in multiple doses, wherein multiple doses can be administered continuously or at specific time intervals. In vitro assays can be used to determine the optimal dose range and/or time plan for administration. In vitro assays to measure the activity of coagulation factors are known in the industry. In addition, the effective dose can be extrapolated from the dose-response curve of an animal model (eg, hemophilia dog) (Mount et al. 2002, Blood 99 (8): 2670).

Intermediate dosages within the above range are also intended to be within the scope of this disclosure. Such doses can be administered to the subject every day, every other day, every week, or any other time schedule determined based on empirical analysis. Exemplary treatments necessitate administration in multiple doses over an extended period of, for example, at least six months.

The lentiviral vectors of the present disclosure can be administered at multiple times. The interval between individual doses can be daily, weekly, monthly or yearly. The interval can also be irregular, as indicated by measuring the blood level of the modified polypeptide or antigen in the patient. The dosage and frequency of the lentiviral vector of the present disclosure vary according to the half-life of the FVIII polypeptide or FIX polypeptide encoded by gene transfer in the patient's body.

The dosage and frequency of administration of the lentiviral vector of the present disclosure can vary depending on whether the treatment is prophylactic or therapeutic. In preventive applications, the composition containing the lentiviral vector of the present disclosure is administered to patients who are not yet in a disease state to enhance the patient's resistance or minimize the impact of the disease. This amount is defined as the "preventive effective dose". A relatively low dose is administered over a long period of time at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives.

The lentiviral vectors of the present disclosure may optionally be administered in combination with other agents that are effective in the treatment of disorders or conditions requiring treatment (eg, prophylactic or therapeutic).

As used herein, the administration of lentiviral vectors of the present disclosure in combination with or in combination with adjuvant therapy means sequential, simultaneous, coexisting, concurrent, concomitant or simultaneous administration or administration of the therapy and the disclosed polypeptide. Those familiar with the art should understand that the administration or administration of the various components in a combination treatment regimen can be timed to enhance the overall effectiveness of the treatment. Based on the selected adjuvant therapy and the teaching content of this manual, those who are familiar with this technology (for example, physicians) can easily identify effective combination treatments without undue experimentation.

It should be further understood that the lentiviral vector of the present disclosure can be combined with or used in combination with one or more agents (for example, to provide a combined treatment regimen). Exemplary agents that can be combined with the lentiviral vectors of the present disclosure include agents that represent the current standard of care for the particular disorder being treated. Such agents can be chemical or biological in nature. The term "biologic" or "biologic agent" refers to any pharmaceutically active agent prepared from living organisms and/or products thereof that is intended to be used as a therapeutic agent.

The amount of the agent to be used in combination with the lentiviral vector of the present disclosure may vary with the subject, or may be administered according to the knowledge in the industry. See, for example, Bruce A Chabner et al., Antineoplastic Agents, in Goodman &Gilman's The Pharmacological Basis of Therapeutics 1233-1287 (Edited by Joel G. Hardman et al., 9th edition 1996). In another embodiment, this agent is administered in an amount that meets the standard of care.

In certain embodiments, the lentiviral vectors of the present disclosure are administered in combination with immunosuppressive agents, anti-allergic agents, or anti-inflammatory agents. These agents generally refer to substances that function to suppress or mask the immune system of the subject treated herein. These agents include substances that inhibit cytokine production, down-regulate or inhibit self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocriptine; danazol; methamphetamine; glutaraldehyde; MHC Anti-idiotypic antibodies to antigens and MHC fragments; cyclosporin A; steroids such as glucocorticoids, such as prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists, including anti-interferon- γ, -β or -α antibody, anti-tumor necrosis factor-α antibody, anti-tumor necrosis factor-β antibody, anti-interleukin-2 antibody and anti-IL-2 receptor antibody; anti-LFA-1 antibody, including anti-CD11a And anti-CD18 antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; full T antibody; soluble peptide containing LFA-3 binding domain; streptokinase; TGF-β; donase; FK506; RS-61443; Oxygualin and rapamycin. In certain embodiments, the agent is an antihistamine. As used herein, an "antihistamine" is an agent that antagonizes the physiological effects of histamine. Examples of antihistamines are chlorpheniramine, diphenhydramine, promethazine, cromolyn, astemizole, azatadine maleate, brompheniramine maleate (Bropheniramine), carbinoxamine maleate, cetirizine hydrochloride (cetirizine), clemastine fumarate (clemastine), cyproheptadine hydrochloride (cyproheptadine), dexbrompheniramine maleate , Dexchloropheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride (diphenhydramine), doxylamine succinate, fexofendadine hydrochloride (fexofendadine), terfeil hydrochloride Terphenadine, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, tripelannamine citrate, tripelennamine hydrochloride, and tripril hydrochloride Pyridine (triprolidine).

Immunosuppressive agents, anti-allergic agents, or anti-inflammatory agents can be incorporated into the lentiviral vector administration regimen. For example, the administration of immunosuppressive agents or anti-inflammatory agents can be started before the administration of the disclosed lentiviral vector, and one or more doses can be continued thereafter. In certain embodiments, immunosuppressive agents or anti-inflammatory agents are administered as prodrugs of lentiviral vectors.

As previously discussed, the lentiviral vector of the present disclosure can be administered in a pharmaceutically effective amount for the in vivo treatment of coagulopathy. In this regard, it should be understood that the lentiviral vectors of the present disclosure can be formulated to facilitate administration and promote the stability of the active agent. Preferably, the pharmaceutical composition according to the present disclosure contains a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer, preservative and the like. Of course, the pharmaceutical composition of the present disclosure can be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the polypeptide.

A variety of tests can be used to evaluate the function of the coagulation system: start partial thromboplastin time (aPTT) test, chromogenic determination, ROTEM ^® determination, prothrombin time (PT) test (also used to determine INR), fibrinogen Test (usually performed by Claus method), platelet count, platelet function test (usually performed by PFA-100), TCT, bleeding time, mixed test (if the patient’s plasma is mixed with normal plasma, is it corrected Abnormal), coagulation factor determination, antiphospholipid antibodies, D-dimer, genetic tests (eg, factor V Leiden, prothrombin mutation G20210A), diluted Russell viper venom time (dRVVT), miscellaneous platelet function tests, coagulation Elastography (TEG or Sonoclot), coagulation elastometry (TEM ^® , such as ROTEM ^® ), or euglobulin solubilization time (ELT).

The aPTT test is a performance indicator that measures the efficacy of both the "intrinsic" (also known as the contact priming pathway) and common coagulation pathways. This test is generally used to measure the coagulation activity of commercially available recombinant coagulation factors (for example, FVIII or FIX). It is used in conjunction with the measurement of the prothrombin time (PT) of the extrinsic pathway.

ROTEM ^® analysis provides information about the overall dynamics of hemostasis: clotting time, clot formation, clot stability and dissolution. The different parameters in the coagulation elasticity assay depend on the activity of the plasma coagulation system, platelet function, fibrinolysis or many factors that affect these interactions. This measurement can provide comprehensive insights into secondary hemostasis.

B.2. Tissue-specific performance

In certain embodiments, it is useful to include one or more miRNA target sequences within the lentiviral vector, the miRNA target sequences being operably linked, for example, with optimized FVIII gene translocation. Therefore, the present disclosure also provides at least one miRNA sequence target that is operably linked to the optimized FVIII or optimized FIX nucleotide sequence or otherwise inserted into the lentiviral vector. The inclusion of more than one copy of the miRNA target sequence in the lentiviral vector can increase the effectiveness of the system.

It also includes different miRNA target sequences. For example, a lentiviral vector that exhibits more than one gene transfer may have gene transfer under the control of more than one miRNA target sequence that may be the same or different. The miRNA target sequence can be tandem, but other arrangements are also included. Gene transfer expression cassettes containing miRNA target sequences can also be inserted into lentiviral vectors in antisense orientation. Antisense targeting can be used to produce viral particles to avoid the expression of gene products that might otherwise be toxic to the production cell.

In other embodiments, the lentiviral vector contains 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence. In certain embodiments, the lentiviral vector does not include any miRNA target sequences. The choice of whether to include miRNA target sequence (and quantity) will be guided by known parameters such as planned tissue target, required performance level, etc.

In one example, the target sequence is the miR-223 target, which has been reported to be the most effective in blocking performance in bone marrow-directed progenitor cells, and at least partially blocking performance in more primitive HSPCs. The miR-223 target can block the expression in differentiated myeloid cells (including granulocytes, monocytes, macrophages, myeloid dendritic cells). The miR-223 target may also be suitable for gene therapy applications that rely on robust gene transfer performance in the lymphoid or red blood cell lineage. The miR-223 target can also block performance very effectively in human HSCs.

In another embodiment, the target sequence is the miR142 target (tccataaagtaggaaacactaca (SEQ ID NO: 7)). In one embodiment, the lentiviral vector contains 4 copies of miR-142 target sequence. In some embodiments, the complementary sequence of hematopoietic-specific microRNA (such as miR-142 (142T)) is incorporated into the 3'untranslated region of the lentiviral vector, so that the transcripts encoding gene transfer are prone to miRNA-mediated Guided downward adjustment. In this way, gene translocation performance can be prevented in hematopoietic lineage antigen presenting cells (APC), while maintaining the gene translocation performance in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose strict post-transcriptional control on gene transfer performance, and thus enable stable delivery and long-term performance of gene transfer. In some embodiments, miR-142 regulation prevents immune-mediated clearance of transduced cells and/or induces antigen-specific regulatory T cells (T regs), and mediates robust immune tolerance to gene-transformed antigens. by.

In some embodiments, the target sequence is the miR181 target. Chen C-Z and Lodish H, Seminars in Immunology (2005) 17(2):155-165 disclosed miR-181, which is a miRNA specifically expressed in B cells in the bone marrow of mice (Chen and Lodish, 2005). The document also discloses that some human miRNAs are related to leukemia.

The target sequence can be fully or partially complementary to the miRNA. The term "fully complementary" means that the nucleic acid sequence of the target sequence is 100% complementary to the sequence of the miRNA that recognizes the target sequence. The term "partially complementary" means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes the target sequence, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, the partially complementary target sequence effectively recognizes the corresponding miRNA, and achieves the prevention or reduction of gene translocation performance in the cell expressing the miRNA. Examples of miRNA target sequences are described in the following documents: WO 2007/000668, WO 2004/094642, WO 2010/055413 or WO 2010/125471, which are incorporated herein by reference in their entirety.

B.3. Heterologous nucleotide sequence

In some embodiments, the isolated nucleic acid molecule further comprises a heterologous nucleotide sequence. In some embodiments, the isolated nucleic acid molecule further comprises at least one heterologous nucleotide sequence. The heterologous nucleotide sequence can be connected to the FVIII or FIX coding sequence of the present disclosure at the 5'end, 3'end, or inserted in the middle. Therefore, in some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is connected to the N-terminus or C-terminus of the FVIII amino acid sequence or the FIX amino acid sequence encoded by the nucleotide sequence, Or insert between the two amino acids in the FVIII amino acid sequence or the FIX amino acid sequence. In some embodiments, the heterologous amino acid sequence can be inserted between two amino acids of the FVIII polypeptide at one or more insertion sites selected from Table 2. In some embodiments, the heterologous amino acid sequence can be inserted into the FVIII polypeptide encoded by the nucleic acid molecule of the present disclosure at any site disclosed in the following documents: International Publication Nos. WO 2013/123457 A1 and WO 2015/106052 A1 Or US Publication No. 2015/0158929 A1, the above-mentioned documents are incorporated herein by reference in their entirety.

In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted into the B domain or a fragment thereof. In some embodiments, the heterologous amino acid sequence is inserted into FVIII immediately downstream of the amino acid corresponding to the amino acid 745 of mature human FVIII (SEQ ID NO: 4). In a specific embodiment, FVIII contains a deletion of amino acids 746-1646 corresponding to mature human FVIII (SEQ ID NO: 4), and the heterologous amino acid sequence encoded by the heterologous nucleotide sequence immediately corresponds to Inserted downstream of the amino acid 745 of mature human FVIII (SEQ ID NO: 4).

Table 2 : Insertion sites of heterologous parts Insertion site Structure domain Insertion site Structure domain Insertion site Structure domain 3 A1 375 A2 1749 A3 18 A1 378 A2 1796 A3 twenty two A1 399 A2 1802 A3 26 A1 403 A2 1827 A3 40 A1 409 A2 1861 A3 60 A1 416 A2 1896 A3 65 A1 442 A2 1900 A3 81 A1 487 A2 1904 A3 116 A1 490 A2 1905 A3 119 A1 494 A2 1910 A3 130 A1 500 A2 1937 A3 188 A1 518 A2 2019 A3 211 A1 599 A2 2068 C1 216 A1 603 A2 2111 C1 220 A1 713 A2 2120 C1 224 A1 745 B 2171 C2 230 A1 1656 a3 area 2188 C2 333 A1 1711 A3 2227 C2 336 A1 1720 A3 2332 CT 339 A1 1725 A3 Note: The insertion site indicates the amino acid position corresponding to the amino acid position of mature human FVIII (SEQ ID NO: 4).

In other embodiments, the isolated nucleic acid molecule further comprises 2, 3, 4, 5, 6, 7, or 8 heterologous nucleotide sequences. In some embodiments, all heterologous nucleotide sequences are the same. In some embodiments, at least one heterologous nucleotide sequence is different from other heterologous nucleotide sequences. In some embodiments, the present disclosure may contain 2, 3, 4, 5, 6, or more than 7 heterologous nucleotide sequences in tandem.

In some embodiments, the heterologous nucleotide sequence encodes an amino acid sequence. In some embodiments, the amino acid sequence encoded by the heterologous nucleotide sequence is a heterologous moiety that can increase the half-life of the FVIII molecule ("half-life extender").

In some embodiments, the heterologous moiety is a peptide or polypeptide with unstructured or structured characteristics that, when incorporated into a protein of the present disclosure, are associated with an increase in half-life in vivo. Non-limiting examples include albumin, albumin fragments, immunoglobulin Fc fragments, human chorionic gonadotropin β subunit C-terminal peptide (CTP), HAP sequence, XTEN sequence, transferrin or fragments thereof, PAS polypeptide, polyglycine linker, polyserine linker, albumin binding portion or any fragment, derivative, variant or combination of these polypeptides. In a specific embodiment, the heterologous amino acid sequence is an immunoglobulin constant region or part thereof, transferrin, albumin, or PAS sequence.

In some aspects, the heterologous moiety includes von Willebrand factor or fragments thereof. In other related aspects, the heterologous moiety may include attachment sites for non-polypeptide moieties (e.g., cysteine amino acid) such as polyethylene glycol (PEG), hydroxyethyl starch ( HES), polysialic acid, or any derivative, variant or combination of these elements. In some aspects, the heterologous moiety comprises a cysteine amino acid used as an attachment site for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), Polysialic acid or any derivative, variant or combination of these elements.

In a specific embodiment, the first heterologous nucleotide sequence encodes the first heterologous part of a half-life extension molecule known in the industry, and the second heterologous nucleotide sequence encodes a half-life extension known in the industry. The second heterologous part of the molecule. In certain embodiments, the first heterologous portion (eg, the first Fc portion) and the second heterologous portion (eg, the second Fc portion) associate with each other to form a dimer. In one embodiment, the second heterologous moiety is a second Fc moiety, wherein the second Fc moiety is linked or associated with the first heterologous moiety (eg, the first Fc moiety). For example, the second heterologous moiety (e.g., the second Fc moiety) can be connected to the first heterologous moiety (e.g., the first Fc moiety) through a linker, or through a covalent or non-covalent bond to the first heterologous moiety. Associate.

In some embodiments, the heterologous moiety is a polypeptide comprising at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, At least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, At least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2500, at least about 3000, or at least about 4000 amino acids, consisting essentially of, or consisting of The amino acid composition.

In other embodiments, the heterologous moiety is a polypeptide comprising about 100 to about 200 amino acids, about 200 to about 300 amino acids, about 300 to about 400 amino acids, about 400 One to about 500 amino acids, about 500 to about 600 amino acids, about 600 to about 700 amino acids, about 700 to about 800 amino acids, about 800 to about 900 amines The base acid, or about 900 to about 1000 amino acids, consists essentially of the amino acid, or consists of the amino acid.

In certain embodiments, the heterologous moiety improves one or more pharmacokinetic properties of the FVIII or FIX protein, but does not significantly affect its biological activity or function.

In certain embodiments, the heterologous moiety increases the in vivo and/or in vitro half-life of the FVIII or FIX protein of the present disclosure. In other embodiments, the heterologous moiety facilitates the visualization or positioning of the FVIII or FIX protein or fragments thereof of the present disclosure (for example, a fragment containing the heterologous moiety after proteolytic cleavage of the FVIII or FIX protein). The visualization and/or positioning of the FVIII or FIX protein or fragments thereof of the present disclosure can be in vivo, in vitro, ex vivo, or a combination thereof.

In other embodiments, the heterologous moiety increases the stability of the FVIII or FIX protein or fragments thereof of the present disclosure (for example, a fragment containing the heterologous moiety after proteolytic cleavage of the FVIII or FIX protein). As used herein, the term "stability" refers to the industry-recognized measure of maintaining one or more physical properties of the FVIII or FIX protein in response to environmental conditions (such as increased or decreased temperature). In certain aspects, the physical property may be maintenance of the covalent structure of the FVIII or FIX protein (for example, the absence of proteolytic cleavage, undesired oxidation, or deamidation). In other aspects, the physical property can also be the presence of FVIII or FIX protein in a correctly folded state (for example, the absence of soluble or insoluble aggregates or precipitates).

In one aspect, the stability of the FVIII or FIX protein is measured by measuring the biophysical properties of the FVIII or FIX protein, such as thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical Function (for example, ability to bind to protein, receptor, or ligand), etc. and/or combinations thereof. On the other hand, the biochemical function is demonstrated by the binding affinity of the interaction. In one aspect, the measure of protein stability is thermal stability, that is, resistance to thermal excitation. Stability can be measured using methods known in the industry, such as HPLC (High Performance Liquid Chromatography), SEC (Size Exclusion Chromatography), DLS (Dynamic Light Scattering), etc. Methods of measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), differential scanning fluorometry (DSF), circular dichroism (CD), and thermal excitation measurement.

In certain aspects, the FVIII or FIX protein encoded by the nucleic acid molecule of the present disclosure contains at least one half-life extender, ie, the in vivo half-life of the corresponding FVIII or FIX protein lacking this heterologous portion increases the in vivo half-life of the FVIII or FIX protein The heterogeneous part. The in vivo half-life of FVIII or FIX protein can be determined by any method known to those skilled in the art, such as activity assay (chromogenic assay or one-step coagulation aPTT assay), ELISA, ROTEM ^™, etc.

In some embodiments, the presence of one or more half-life extenders results in an increase in the half-life of the FVIII or FIX protein compared to the half-life of the corresponding protein lacking the one or more half-life extenders. The half-life of a FVIII or FIX protein comprising a half-life extender is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times.

In one embodiment, the half-life of a FVIII or FIX protein comprising a half-life extender is about 1.5 times to about 20 times, about 1.5 times to about 15 times, or about 1.5 times longer than the in vivo half-life of the corresponding protein lacking such a half-life extender To about 10 times. In another embodiment, the half-life of a FVIII or FIX protein comprising a half-life extender is increased by about 2-fold to about 10-fold, or about 2-fold to about 9-fold compared to the in vivo half-life of a corresponding protein lacking such a half-life extender , About 2 times to about 8 times, about 2 times to about 7 times, about 2 times to about 6 times, about 2 times to about 5 times, about 2 times to about 4 times, about 2 times to about 3 times, about 2.5 times to about 10 times, about 2.5 times to about 9 times, about 2.5 times to about 8 times, about 2.5 times to about 7 times, about 2.5 times to about 6 times, about 2.5 times to about 5 times, about 2.5 times To about 4 times, about 2.5 times to about 3 times, about 3 times to about 10 times, about 3 times to about 9 times, about 3 times to about 8 times, about 3 times to about 7 times, about 3 times to about 6 times, about 3 times to about 5 times, about 3 times to about 4 times, about 4 times to about 6 times, about 5 times to about 7 times, or about 6 times to about 8 times.

In other embodiments, the half-life of the FVIII or FIX protein comprising the half-life extender is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

In still other embodiments, the half-life of the FVIII or FIX protein comprising the half-life extender is about 15 hours to about 2 weeks, about 16 hours to about 1 week, about 17 hours to about 1 week, about 18 hours to about 1 week , About 19 hours to about 1 week, about 20 hours to about 1 week, about 21 hours to about 1 week, about 22 hours to about 1 week, about 23 hours to about 1 week, about 24 hours to about 1 week, about 36 hours to about 1 week, about 48 hours to about 1 week, about 60 hours to about 1 week, about 24 hours to about 6 days, about 24 hours to about 5 days, about 24 hours to about 4 days, about 24 hours To about 3 days, or about 24 hours to about 2 days.

In some embodiments, the average half-life of each subject of the FVIII or FIX protein comprising the half-life extender is about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours , About 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about 6 days, about 7 days (1 week), about 8 days, about 9 days, about 10 days, about 11 days , About 12 days, about 13 days, or about 14 days.

One or more half-life extenders can be fused to the C-terminus or N-terminus of FVIII or FIX or inserted into FVIII or FIX.

B.3.a. Immunoglobulin constant region or part thereof

In another aspect, the heterologous moiety comprises one or more immunoglobulin constant regions or portions thereof (eg, Fc region). In one embodiment, the isolated nucleic acid molecule of the present disclosure further comprises a heterologous nucleic acid sequence encoding an immunoglobulin constant region or part thereof. In some embodiments, the immunoglobulin constant region or part thereof is an Fc region.

The immunoglobulin constant region is composed of domains denoted as CH (constant weight) domains (CH1, CH2, etc.). Depending on the isotype (ie, IgG, IgM, IgA, IgD, or IgE), the constant region can be composed of 3 or 4 CH domains. Some isotypes (such as IgG) constant regions also contain hinge regions. See Janeway et al. 2001, Immunobiology , Garland Publishing, New York, New York.

The immunoglobulin constant regions or portions thereof used to produce the FVIII protein of the present disclosure can be obtained from a variety of different sources. In one embodiment, the immunoglobulin constant region or part thereof is derived from human immunoglobulin. However, it should be understood that the immunoglobulin constant region or part thereof may be derived from an immunoglobulin of another mammalian species including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs). ) Or non-human primate (e.g., chimpanzee, macaque) species. In addition, immunoglobulin constant regions or parts thereof can be derived from any immunoglobulin class (including IgM, IgG, IgD, IgA, and IgE) and any immunoglobulin isotype (including IgG1, IgG2, IgG3, and IgG4). In one embodiment, the human isotype IgG1 is used.

Multiple immunoglobulin constant region gene sequences (for example, human constant region gene sequences) can be obtained in the form of publicly available deposits. It is possible to select constant region domain sequences that have specific effector functions (or lack specific effector functions) or have specific modifications to reduce immunogenicity. The sequences of many antibodies and antibody-encoding genes have been disclosed and suitable Ig constant region sequences (for example, hinge, CH2 and/or CH3 sequences or parts thereof) can be derived from these sequences using industry-recognized techniques. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain the polypeptides of the present disclosure. It should be further understood that the scope of the present disclosure encompasses alleles, variants, and mutations of the constant region DNA sequence.

The sequence of the immunoglobulin constant region or part thereof can be cloned, for example, using polymerase chain reaction and primers selected to amplify the domain of interest. In order to clone the sequence of the immunoglobulin constant region or part thereof from the antibody, mRNA can be isolated from hybridoma, spleen or lymphocyte, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. The PCR amplification method is described in detail in the following documents: U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, "PCR Protocols: A Guide to Methods and Applications" Edited by Innis et al., Academic Press, San Diego, California (1990 ); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated by consensus constant region primers or by more specific primers based on the disclosed heavy and light chain DNA and amino acid sequences. PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, the library can be screened by consensus primers or larger homologous probes (such as mouse constant region probes). Many primer sets suitable for amplifying antibody genes are known in the industry (for example, 5'primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends ( Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequence (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). The cloning of the antibody sequence is further described by Newman et al. in 1995 In U.S. Patent No. 5,658,570 filed on January 25, 2005, the patent is incorporated herein by reference.

The immunoglobulin constant region used herein may include all domains and hinge regions or parts thereof. In one embodiment, the immunoglobulin constant region or part thereof comprises a CH2 domain, a CH3 domain and a hinge region, that is, an Fc region or an FcRn binding partner.

As used herein, the term "Fc region" is defined as the portion of a polypeptide that corresponds to the Fc region of natural Ig, that is, as formed by the dimeric association of the corresponding Fc domains of its two heavy chains. The natural Fc region forms a homodimer with another Fc region. In contrast, the term "gene-fused Fc region" or "single-chain Fc region" (scFc region) as used herein refers to a synthetic dimeric Fc region that is genetically linked within a single polypeptide chain (ie, encoded in In a single continuous gene sequence) the Fc domain constitutes. See International Publication No. WO 2012/006635, which is incorporated herein by reference in its entirety.

In one embodiment, the "Fc region" refers to the following part of a single Ig heavy chain, which starts in the hinge region immediately adjacent to the papain cleavage site (ie residue 216 in IgG, and the first heavy chain constant region Residue 114) upstream, and finally the C-terminus of the antibody. Therefore, the complete Fc region contains at least the hinge domain, the CH2 domain and the CH3 domain.

The immunoglobulin constant region or part thereof may be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal cavity, lung airway, nasal surface, vaginal surface, colon and rectal surface (US Patent No. 6,485,726). The FcRn binding partner is the part of the immunoglobulin that binds to FcRn.

The FcRn receptor has been isolated from several mammalian species including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG at relatively low pH (but does not bind to other immunoglobulin classes, such as IgA, IgM, IgD, and IgE), and actively transports IgG across cells in the direction from the lumen to the serosal membrane. IgG is released at high pH. It is expressed in adult epithelial tissues (US Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834; US 2003-0235536A1), which includes lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), Tubular epithelium at the proximal end of the kidney (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as the nasal epithelium, vaginal surface, and bile duct tree surface.

The FcRn binding partners that can be used in the present disclosure encompass molecules that can specifically bind to the FcRn receptor, including intact IgG, Fc fragments of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region in the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The main contact area between Fc and FcRn is close to the junction of CH2 and CH3 domains. The Fc-FcRn contacts are all located within a single Ig heavy chain. FcRn binding partners include intact IgG, Fc fragments of IgG, and other IgG fragments of IgG that include the complete binding region of FcRn. The main contact sites include the amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and the amino acid residues 385-387, 428 of the CH3 domain And 433-436. References to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda, Maryland.

FcRn can effectively shuttle across the epithelial barrier to bind to the Fc region of FcRn or the FcRn binding partner, thereby providing a non-invasive means for systemic administration of the desired therapeutic molecules. In addition, a fusion protein containing an Fc region or an FcRn binding partner is endocytosed by cells expressing FcRn. However, these fusion proteins are not labeled for degradation, but are recycled into the circulation again, thereby increasing the in vivo half-life of these proteins. In certain embodiments, the part of the immunoglobulin constant region is an Fc region or an FcRn binding partner, which usually associates with another Fc region or another FcRn binding partner through disulfide bonds and other non-specific interactions , To form dimers and higher order multimers.

Two FcRn receptors can bind a single Fc molecule. Crystallographic data indicate that each FcRn molecule binds to a single polypeptide of the Fc homodimer. In one embodiment, the FcRn binding partner (eg, the Fc fragment of IgG) is linked to a biologically active molecule to provide oral, buccal, sublingual, transrectal, transvaginal, nasal or pulmonary route administration. Sol, or a means of delivering biologically active molecules via the ocular route. In another embodiment, the FVIII protein can be administered invasively, such as subcutaneously or intravenously.

The FcRn binding partner region is a molecule or part thereof that can be specifically bound by the FcRn receptor and then actively transported through the FcRn receptor of the Fc region. Specific binding means that two molecules form a relatively stable complex under physiological conditions. Specific binding is characterized by high affinity and low to medium capacity. In contrast, non-specific binding usually has low affinity and medium to high capacity. Generally, when the affinity constant KA is higher than 10 ⁶ M ^-1 or higher than 10 ⁸ M ^-1 , the binding is considered specific. If necessary, the binding conditions can be changed to reduce non-specific binding without substantially affecting specific binding. Those skilled in the art can use conventional techniques to optimize appropriate binding conditions, such as molecular concentration, ionic strength of the solution, temperature, time allowed for binding, concentration of blocking agents (for example, serum albumin, casein), etc.

In certain embodiments, the FVIII protein encoded by the nucleic acid molecule of the present disclosure contains one or more truncated Fc regions, but it is sufficient to impart Fc receptor (FcR) binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn (ie, the FcRn-binding portion) contains approximately amino acids 282-438 of IgG1, EU numbering (main contact sites are amino acids 248, 250-257, 272 of CH2 domain) , 285, 288, 290-291, 308-311 and 314 and the amino acid residues of the CH3 domain 385-387, 428 and 433-436). Therefore, the Fc region of the present disclosure may comprise or consist of the FcRn binding portion. The FcRn binding portion can be derived from the heavy chain of any isotype (including IgG1, IgG2, IgG3, and IgG4). In one example, the FcRn binding portion from an antibody of the human isotype IgG1 is used. In another example, the FcRn binding portion from an antibody of the human isotype IgG4 is used.

The Fc region can be obtained from a variety of different sources. In one embodiment, the Fc region of the polypeptide is derived from human immunoglobulin. However, it should be understood that the Fc portion may be derived from an immunoglobulin of another mammalian species including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates. Animal-like (for example, chimpanzee, macaque) species. In addition, the polypeptides of the Fc domain or part thereof can be derived from any immunoglobulin class (including IgM, IgG, IgD, IgA, and IgE) and any immunoglobulin isotype (including IgG1, IgG2, IgG3, and IgG4). In another embodiment, the human isotype IgG1 is used.

In certain embodiments, the Fc variant confers at least one change in effector function conferred by the Fc portion comprising the wild-type Fc domain (e.g., the Fc region binds to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) Or complement proteins (eg, C1q) or the ability to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC) to improve or decrease). In other embodiments, the Fc variant provides engineered cysteine residues.

The Fc region of the present disclosure can utilize industry-recognized Fc variants, which are known to confer effector functions and/or changes (for example, enhancement or reduction) in FcR or FcRn binding. Specifically, the Fc region of the present disclosure may include, for example, changes (for example, substitutions) at one or more amino acid positions disclosed in the following documents: International PCT Publications WO 88/07089A1, WO 96/14339A1, WO 98/05787A1, WO 98/23289A1, WO 99/51642A1, WO 99/58572A1, WO 00/09560A2, WO 00/32767A1, WO 00/42072A2, WO 02/44215A2, WO 02/060919A2, WO 03/074569A2 WO 04/016750A2, WO 04/029207A2, WO 04/035752A2, WO 04/063351A2, WO 04/074455A2, WO 04/099249A2, WO 05/040217A2, WO 04/044859, WO 05/070963A1, WO 05/077981A2 WO 05/092925A2, WO 05/123780A2, WO 06/019447A1, WO 06/047350A2, and WO 06/085967A2; US Patent Publication Nos. US 2007/0231329, US 2007/0231329, US 2007/0237765, US 2007/0237766, US 2007/0237767, US 2007/0243188, US 2007/0248603, US 2007/0286859, US 2008/0057056; or US Patent 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375, 6,528,624; ; 6,821,505; 6,998,253; 7,083,784; 7,404,956 and 7,317,091, each of which is incorporated herein by reference. In one embodiment, specific changes can be made at one or more disclosed amino acid positions (for example, specific substitutions of one or more amino acids disclosed in the industry). In another embodiment, different changes can be made at one or more of the disclosed amino acid positions (for example, different substitutions of one or more amino acid positions disclosed in the industry).

The Fc region or FcRn binding partner of IgG can be modified according to accepted procedures (such as site-directed mutagenesis, etc.) to produce a modified IgG or Fc fragment or part thereof that will be bound by FcRn. Such modifications include modifications away from the FcRn contact site as well as modifications within the contact site that retain or even enhance binding to FcRn. For example, the following single amino acid residues in human IgG1 Fc (Fcγ1) can be substituted without significantly losing the binding affinity of Fc to FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A , D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, Y300F , V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, , E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, KS392A, L398A, , D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where, for example, P238A represents the wild type substituted by alanine at position number 238 Type proline. As an example, a specific example incorporates the N297A mutation to remove the highly conserved N glycosylation site. In addition to alanine, other amino acids can be substituted for wild-type amino acids at the positions specified above. Mutations can be introduced into the Fc alone, resulting in more than one hundred Fc regions that differ from the natural Fc. In addition, a combination of two, three or more of these individual mutations can be introduced together, resulting in hundreds of additional Fc regions.

Some of the above mutations can confer new functions to the Fc region or FcRn binding partner. For example, one example incorporates N297A, thereby removing the highly conserved N glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the Fc region, and preventing the Fc region from binding to FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA, and does not damage the affinity for FcRn (Routledge et al. 1995, Transplantation 60: 847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As another example of the new function resulting from the above-mentioned mutations, the affinity for FcRn may be increased, in some cases exceeding that of the wild-type. This increased affinity can reflect an increased "association" rate, a decreased "dissociation" rate, or both an increased "association" rate and a decreased "dissociation" rate. Examples of mutations believed to confer increased affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

In addition, at least three human Fcγ receptors appear to recognize the binding sites on the lower hinge region of IgG, usually amino acids 234-237. Therefore, for example, by replacing the amino acids 233-236 "ELLG" (SEQ ID NO: 8) of human IgG1 with the corresponding sequence "PVA" (one amino acid deletion) from IgG2, mutations in this region can be generated. Another example of new features and possible reduced immunogenicity. It has been shown that when such mutations have been introduced, FcyRI, FcyRII, and FcyRIII, which mediate multiple effector functions, will not bind to IgG1. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

In another embodiment, the immunoglobulin constant region or part thereof comprises an amino acid sequence in the hinge region or part thereof that forms one or more disulfide bonds with the second immunoglobulin constant region or part thereof. The second immunoglobulin constant region or part thereof can be linked to the second polypeptide, so that the FVIII protein and the second polypeptide are bound together. In some embodiments, the second polypeptide is an enhancer moiety. As used herein, the term "enhancer portion" refers to a molecule, fragment or polypeptide component capable of enhancing the procoagulant activity of FVIII. The enhancer portion can be a cofactor, such as soluble tissue factor (sTF) or procoagulant peptide. Therefore, after starting FVIII, the enhancer part can be used to enhance FVIII activity.

In certain embodiments, the FVIII protein encoded by the nucleic acid molecule of the present disclosure contains amino acid substitutions (eg, Fc variants) to the immunoglobulin constant region or part thereof, which amino acid substitutions change the Ig constant region The antigen-independent effector function, especially the circulating half-life of the protein.

B.3.b. scFc region

In another aspect, the heterologous portion comprises a scFc (single chain Fc) region. In one embodiment, the isolated nucleic acid molecule of the present disclosure further comprises a heterologous nucleic acid sequence encoding the scFc region. The scFc region includes at least two immunoglobulin constant regions or parts thereof (for example, Fc parts or domains (for example, 2, 3, 4, 5, 6 or more Fc parts or domains) within the same linear polypeptide chain )), the constant region or part can be folded (for example, intramolecular or intermolecular folding) to form a functional scFc region connected by an Fc peptide linker. For example, in one embodiment, the polypeptide of the present disclosure can bind to at least one Fc receptor (eg, FcRn, FcγR receptor (eg, FcγRIII), or complement protein (eg, C1q)) via its scFc region to improve Half-life may trigger immune effector functions (eg, antibody-dependent cytotoxicity (ADCC), phagocytosis or complement-dependent cytotoxicity (CDCC) and/or improved manufacturability).

B.3.c.CTP

In another aspect, the heterologous moiety comprises a C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin or a fragment, variant or derivative thereof. It is known that one or more CTP peptides inserted into a recombinant protein increase the half-life of the protein in vivo. See, for example, U.S. Patent No. 5,712,122, which is incorporated herein by reference in its entirety.

Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 9) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 10). See, for example, U.S. Patent Application Publication No. US 2009/0087411 A1, which is incorporated by reference.

B.3.d. XTEN sequence

In some embodiments, the heterologous portion comprises one or more XTEN sequences, fragments, variants or derivatives thereof. As used herein, "XTEN sequence" refers to a polypeptide having an extended length of a non-naturally occurring substantially non-repetitive sequence, which is mainly composed of small hydrophilic amino acids, and the sequence has a relatively low degree under physiological conditions. Or do not have a secondary or tertiary structure. As a heterologous part, XTEN can be used as a half-life extension part. In addition, XTEN can provide desired properties, including but not limited to enhanced pharmacokinetic parameters and solubility characteristics.

Incorporating a heterologous portion comprising an XTEN sequence into a protein of the present disclosure can impart one or more of the following advantageous properties to the protein: conformational flexibility, enhanced water solubility, high degree of protease resistance, low immunogenicity, and mammalian Low binding or increased hydrodynamic (or Stokes) radius of the receptor.

In certain aspects, XTEN sequences can increase pharmacokinetic properties, such as longer in vivo half-life or increased area under the curve (AUC), so that compared with the same protein but without a heterologous portion of XTEN, the present disclosure The protein stays in the body for an increased period of time and has procoagulant activity.

In some embodiments, XTEN sequences that can be used in the present disclosure are those having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, A peptide or polypeptide with 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid residues. In certain embodiments, XTEN has greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, Greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 Residues, or peptides or polypeptides of greater than 120 to about 400 residues. In a specific embodiment, XTEN comprises an amino acid sequence longer than 42 amino acids and shorter than 144 amino acids.

The XTEN sequence of the present disclosure may contain one or more sequence motifs of 5 to 14 (for example, 9 to 14) amino acid residues or at least 80%, 90%, 91%, 92% of the sequence motifs. %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, wherein the motif contains 4 to 6 types selected from glycine (G), The amino acids of alanine (A), serine (S), threonine (T), glutamine (E) and proline (P) (for example, 5 kinds of amino acids) are basically composed of The amino acid consists of or consists of the amino acid. See US 2010-0239554 A1.

In some embodiments, XTEN comprises non-overlapping sequence motifs, of which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94% , Or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units selected from non-overlapping sequences of a single motif family selected from Table 3 Composition to get the family sequence.

As used herein, "family" means that XTEN has a motif selected from only a single motif category in Table 3; that is, AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and the XTEN is not selected Any other amino acids from the family motif to achieve the desired characteristics, such as allowing the incorporation of restriction sites by encoding nucleotides, incorporation of cleavage sequences, or to achieve better linkage to FVIII. In some embodiments of the XTEN family, the XTEN sequence includes the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family Or multiple units of non-overlapping sequence motifs of the BD family, the resulting XTEN exhibits the above-mentioned range of homology. In other embodiments, XTEN comprises multiple units of motif sequences from two or more motif families in Table 3.

These sequences can be selected to achieve the required physical/chemical characteristics, including characteristics such as net charge, hydrophilicity, lack of secondary structure, or lack of repetition. These characteristics are given by the amino acid composition of the motif, which will be more comprehensively described below. describe. In the above examples in this paragraph, the methods described herein can be used to select and assemble motifs incorporated into XTEN to achieve XTEN with about 36 to about 3000 amino acid residues.

Table 3. XTEN sequence motifs and motif families of 12 amino acids Motif family * Motif sequence SEQ ID NO: AD GESPGGSSGSES 11 AD GSEGSSGPGESS 12 AD GSSESGSSEGGP 13 AD GSGGEPSESGSS 14 AE, AM GSPAGSPTSTEE 15 AE, AM, AQ GSEPATSGSETP 16 AE, AM, AQ GTSESATPESGP 17 AE, AM, AQ GTSTEPSEGSAP 18 AF, AM GSTSESPSGTAP 19 AF, AM GTSTPESGSASP 20 AF, AM GTSPSGESSTAP twenty one AF, AM GSTSSTAESPGP twenty two AG, AM GTPGSGTASSSP twenty three AG, AM GSSTPSGATGSP twenty four AG, AM GSSPSASTGTGP 25 AG, AM GASPGTSSTGSP 26 AQ GEPAGSPTSTSE 27 AQ GTGEPSSTPASE 28 AQ GSGPSTESAPTE 29 AQ GSETPSGPSETA 30 AQ GPSETSTSEPGA 31 AQ GSPSEPTEGTSA 32 BC GSGASEPTSTEP 33 BC GSEPATSGTEPS 34 BC GTSEPSTSEPGA 35 BC GTSTEPSEPGSA 36 BD GSTAGSETSTEA 37 BD GSETATSGSETA 38 BD GTSESATSESGA 39 BD GTSTEASEGSAS 40 * Indicates a single motif sequence, which when used together in various permutations to obtain a "family sequence"

Examples of XTEN sequences that can be used as heterologous parts in the chimeric protein of the present disclosure are disclosed in, for example, the following documents: U.S. Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1 , 2011/0077199 A1 or 2011/0172146 A1, or International Patent Publication No. WO 2010/091122 A1, WO 2010/144502 A2, WO 2010/144508 A1, WO 2011/028228 A1, WO 2011/028229 A1 or WO 2011/028344 A2. Incorporate each document in its entirety by reference.

XTEN can have different lengths for insertion into or connection to FVIII. In one embodiment, the length of one or more XTEN sequences is selected based on the characteristics or functions to be achieved in the fusion protein. Depending on the expected characteristics or function, XTEN can be a short sequence or a medium length sequence or a longer sequence that can serve as a carrier. In certain embodiments, XTEN includes short segments of about 6 to about 99 amino acid residues, a medium length of about 100 to about 399 amino acid residues, and about 400 to about 1000 and up to about 3000. The longer length of an amino acid residue. Therefore, the XTEN inserted into or connected to FVIII may have about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about A length of 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues. In other embodiments, the length of the XTEN sequence is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, About 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues.

The exact length of XTEN inserted into or linked to FVIII can be varied without adversely affecting the activity of FVIII. In one embodiment, the length of one or more XTEN used herein is 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amines. The base acid can be selected from one or more XTEN family sequences; the family is AD, AE, AF, AG, AM, AQ, BC or BD.

In some embodiments, the XTEN sequence used in the present disclosure is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99% or 100% same: AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, AG2004 and any combination thereof. See US 2010-0239554 A1. In a specific embodiment, XTEN comprises AE42, AE72, AE144, AE288, AE576, AE864, AG 42, AG72, AG144, AG288, AG576, AG864, or any combination thereof.

Exemplary XTEN sequences that can be used as the heterologous portion in the chimeric protein of the present disclosure include XTEN AE42-4 (SEQ ID NO: 41), XTEN 144-2A (SEQ ID NO: 42), XTEN A144-3B ( SEQ ID NO: 43), XTEN AE144-4A (SEQ ID NO: 44), XTEN AE144-5A (SEQ ID NO: 45), XTEN AE144-6B (SEQ ID NO: 46), XTEN AG144-1 (SEQ ID NO: 47), XTEN AG144-A (SEQ ID NO: 48), XTEN AG144-B (SEQ ID NO: 49), XTEN AG144-C (SEQ ID NO: 50) and XTEN AG144-F (SEQ ID NO: 51). In a specific embodiment, XTEN is encoded by SEQ ID NO:52.

In some embodiments, less than 100% of the amino acids of XTEN are selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), and glutamine (E) And proline (P), or less than 100% of the sequence consists of the sequence motif from Table 3 or the XTEN sequence provided herein. In such embodiments, the remaining amino acid residues of XTEN are selected from any of the other 14 natural L-amino acids, but can be preferably selected from hydrophilic amino acids, so that the XTEN sequence contains at least about 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% hydrophilic amino acids.

The content of hydrophobic amino acid in XTEN utilized in the conjugation construct may be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. Hydrophobic residues that are less favorable in the construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, oleucine, and methionine. In addition, the XTEN sequence may contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: methionine (for example, to avoid oxidation), or aspartic acid And glutamic acid (to avoid deamidation).

The one or more XTEN sequences may be inserted into the C-terminus or N-terminus of the amino acid sequence encoded by the nucleotide sequence, or inserted into one of the two amino acids in the amino acid sequence encoded by the nucleotide sequence. between. For example, XTEN can be inserted between two amino acids at one or more insertion sites selected from Table 2. Examples of sites within FVIII that allow XTEN insertion can be found in, for example, International Publication No. WO 2013/123457 A1 or U.S. Publication No. 2015/0158929 A1, which is incorporated herein by reference in its entirety.

B.3.e. Albumin or its fragments, derivatives or variants

In some embodiments, the heterologous moiety comprises albumin or a functional fragment thereof. Human serum albumin (HSA, or HA) is a full-length protein with 609 amino acids, responsible for most of the serum osmotic pressure, and also acts as a carrier for endogenous and exogenous ligands. The term "albumin" as used herein includes full-length albumin or functional fragments, variants, derivatives or analogs thereof. Examples of albumin or fragments or variants thereof are disclosed in the following documents: US Patent Publication No. 2008/0194481A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1 or 2008/0153751 A1 or PCT Application Publication No. WO 2008 /033413 A2, WO 2009/058322 A1 or WO 2007/021494 A2, which is incorporated herein by reference in its entirety.

In one embodiment, the FVIII protein encoded by the nucleotide molecule of the present disclosure comprises albumin, fragments or variants thereof further linked to a second heterologous moiety, the second heterologous moiety selected from immunoglobulin constant Region or part thereof (eg, Fc region), PAS sequence, HES, and PEG.

B.3.f. Albumin binding part

In certain embodiments, the heterologous moiety is an albumin binding moiety, which comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin binding antibody fragment, or any combination thereof.

For example, the albumin binding protein can be a bacterial albumin binding protein, antibody or antibody fragment, including domain antibodies (see US Patent No. 6,696,245). For example, the albumin binding protein may be a bacterial albumin binding domain, such as a streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-83). Other examples of albumin binding peptides that can be used as conjugation partners are, for example, those albumin binding peptides having the consensus sequence of _{Cys-Xaa 1} -Xaa ₂ -Xaa ₃ -Xaa _{4 -Cys (SEQ ID NO: 52), wherein} Xaa ₁ is Asp, Asn, Ser, Thr, or Trp; Xaa ₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa ₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa ₄ is Asp, Gly, Leu, Phe, Ser, or Thr, as described in US Patent Application Publication No. 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

Domain 3 from Streptococcus protein G (as disclosed by Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002)) is bacterial albumin Examples of binding domains. Examples of albumin binding peptides include a series of peptides having the core sequence DICLPRWGCLW (SEQ ID NO: 54). See, for example, Dennis et al., J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples of albumin binding antibody fragments are disclosed in the following documents: Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al., Cancer Immunol.Immunother. 56:303-317 (2007) ); and Holt et al., Prot. Eng. Design Sci ., 21:283-288 (2008), which is incorporated herein by reference in its entirety. An example of such an albumin binding moiety is 2-(3-maleiminopropylamino)-6-(4-(4-iodophenyl)butyramido)hexanoate ("Albu" Label), as disclosed in Trussel et al., Bioconjugate Chem. 20:2286-2292 (2009).

Fatty acids, particularly long-chain fatty acids (LCFA) and long-chain fatty acid-like albumin binding compounds can be used to extend the in vivo half-life of the FVIII protein of the present disclosure. An example of an LCFA-like albumin binding compound is 16-(1-(3-(9-(((2,5-dioxopyrrolidin-1-yloxy)carbonyloxy)-methyl)-7- Sulfo-9H-fluoren-2-ylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio)hexadecanoic acid (see, for example, WO 2010/140148 ).

B.3.g. PAS sequence

In other embodiments, the heterologous moiety is a PAS sequence. The PAS sequence as used herein means an amino acid sequence mainly comprising alanine and serine residues or mainly comprising alanine, serine and proline residues, which are formed under physiological conditions Random spiral conformation. Therefore, the PAS sequence is a building block, amino acid polymer or sequence box consisting of alanine, serine and proline, consisting essentially of or consisting of the amino acid, which can be used As part of the heterologous part of the chimeric protein. However, those skilled in the art know that amino acid polymers can also form random helical conformations when residues other than alanine, serine and proline are added as minor components in the PAS sequence.

The term "minor component" as used herein means that amino acids other than alanine, serine and proline can be added to the PAS sequence to a certain degree, for example, up to about 12%, that is, the PAS sequence Up to about 12 of 100 amino acids in the PAS sequence; up to about 10%, that is, about 10 of the 100 amino acids in the PAS sequence; up to about 9%, that is, about 9 of the 100 amino acids; up to About 8%, that is about 8 out of 100 amino acids; about 6%, that is about 6 out of 100 amino acids; about 5%, that is about 5 out of 100 amino acids; about 4 %, that is about 4 out of 100 amino acids; about 3%, that is about 3 out of 100 amino acids; about 2%, that is about 2 out of 100 amino acids; about 1%, That is, about 1 in 100 amino acids. Amino acids other than alanine, serine and proline can be selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr , And Val.

Under physiological conditions, the PAS sequence extensions form a random helical conformation, which can mediate the increased in vivo and/or in vitro stability of the FVIII protein. Since the random helix domain itself does not have a stable structure or function, it basically retains the biological activity mediated by the FVIII protein. In other embodiments, the PAS sequence forming the random helical domain is biologically inert, especially with regard to proteolysis in plasma, immunogenicity, isoelectric point/electrostatic behavior, binding or internalization with cell surface receptors, but still It is biodegradable, which provides a clear advantage over synthetic polymers such as PEG.

Non-limiting examples of PAS sequences forming a random helical conformation include amino acid sequences selected from: ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 55), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 56), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 57), APSSPSPSAPSSPSPASPS (SEQ ID NO: 58), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 59), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 60) and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 61) or any combination thereof. Additional examples of PAS sequences can be found in the following documents: for example, U.S. Patent Publication No. 2010/0292130 A1 and PCT Application Publication No. WO 2008/155134 A1.

B.3.h.HAP sequence

In certain embodiments, the heterologous moiety is a homoamino acid polymer (HAP) rich in glycine. The HAP sequence may include a repeating sequence of glycine, the length of the repeating sequence is at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids. In one embodiment, the HAP sequence can extend the half-life of the portion fused to or linked to the HAP sequence. Non-limiting examples of HAP sequences include but are not limited to (Gly) _n , (Gly ₄ Ser) _n or S(Gly ₄ Ser) _n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

B.3.i. Transferrin or its fragments

In certain embodiments, the heterologous moiety is transferrin or fragments thereof. Any transferrin can be used to prepare the FVIII protein of the present disclosure. As an example, wild-type human TF (TF) is a protein with 679 amino acids, approximately 75 KDa (excluding glycosylation), and has two main domains N (approximately 330 amino acids) that appear to be derived from gene duplication. And C (about 340 amino acids). See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847, and S95936 (www.ncbi.nlm.nih.gov/), all of which are incorporated herein by reference in their entirety. Transferrin contains two domains, N domain and C domain. The N domain contains two subdomains, namely the N1 domain and the N2 domain, and the C domain contains two subdomains, namely the C1 domain and the C2 domain.

In one embodiment, the heterologous portion of transferrin includes a transferrin splice variant. In one example, the transferrin splice variant may be a splice variant of human transferrin, such as Genbank accession number AAA61140. In another embodiment, the transferrin portion of the chimeric protein includes one or more domains of the transferrin sequence, such as N domain, C domain, N1 domain, N2 domain, C1 domain, C2 Domain or any combination thereof.

B.3.j. Removal of receptors

In certain embodiments, the heterologous moiety is a scavenging receptor, fragment, variant or derivative thereof. LRP1 is a 600 kDa membrane intrinsic protein, which is involved in receptor-mediated clearance of a variety of proteins (such as factor X). See, for example, Narita et al., Blood 91:555-560 (1998).

B.3.k. Von Willebrand Factor or its fragments

In certain embodiments, the heterologous moiety is von Willebrand factor (VWF) or one or more fragments thereof.

VWF (also known as F8VWF) is a polyglycan protein that exists in plasma, and is found in the endothelium (in Weibel-Palade body), megakaryocytes (alpha particles of platelets) and endothelial cells. Constitutively produced in the subcutaneous connective tissue. The basic VWF monomer is a protein with 2813 amino acids. Each monomer contains multiple specific domains with specific functions, namely D'and D3 domains (which together bind to factor VIII), A1 domains (which bind to platelet GPIb-receptors, heparin and/or possibly Collagen), A3 domain (which binds to collagen), C1 domain (where the RGD domain binds to platelet integrin αIIbβ3 when activated), and the "cysteine knot" domain at the C-terminus of the protein (its It is shared by VWF and platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ) and β-human chorionic gonadotropin (βHCG)).

The 2813 monomer amino acid sequence of human VWF is reported as the accession number NP000543.2 in Genbank. The nucleotide sequence encoding human VWF is reported as the accession number NM000552.3 in Genbank. SEQ ID NO: 62 is the amino acid sequence reported in Genbank Accession No. NM000552.3. The D'domain includes amino acids 764 to 866 of SEQ ID NO: 62. The D3 domain includes amino acids 867 to 1240 of SEQ ID NO: 62.

In plasma, 95%-98% of FVIII circulates as a tight non-covalent complex with full-length VWF. The formation of this complex is important for maintaining proper FVIIII plasma levels in the body. Lenting et al., Blood. 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7): 1353-60 (2007). When FVIII is activated due to proteolysis at positions 372 and 740 in the heavy chain and position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII.

In certain embodiments, the heterologous moiety is full-length von Willebrand factor. In other embodiments, the heterologous moiety is a von Willebrand factor fragment. As used herein, the term "VWF fragment" or "VWF fragments" as used herein refers to any VWF fragment that interacts with FVIII and retains at least one or more of those that are normally provided by full-length VWF to FVIII Properties, such as preventing premature initiation of FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes (which can lead to premature clearance), preventing clearance of FVIII that can bind to naked FVIII instead of VWF The receptor binds and/or stabilizes FVIII heavy chain and light chain interactions. In a specific embodiment, the heterologous moiety is a (VWF) fragment comprising the D'domain and the D3 domain of VWF. The VWF fragment comprising the D'domain and the D3 domain may also comprise a VWF domain selected from: A1 domain, A2 domain, A3 domain, D1 domain, D2 domain, D4 domain, B1 domain , B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. Other examples of polypeptides with FVIII activity fused to the VWF fragment are disclosed in the following documents: U.S. Provisional Patent Application No. 61/667,901 and U.S. Publication No. 2015/0023959 A1 filed on July 3, 2012, both of which are It is incorporated herein by reference in its entirety.

B.3.l. Connecting sub-part

In certain embodiments, the heterologous moiety is a peptide linker.

As used herein, the term "peptide linker" or "linker portion" refers to a peptide or polypeptide sequence (for example, a synthetic peptide or polypeptide sequence) that connects two domains in a linear amino acid sequence of a polypeptide chain.

In some embodiments, a heterologous nucleotide sequence encoding a peptide linker can be inserted into the optimized FVIII polynucleotide sequence of the present disclosure and a heterologous nucleotide encoding, for example, one of the aforementioned heterologous parts (such as albumin). Between sequences. The peptide linker can provide flexibility to the chimeric polypeptide molecule. The linker is usually not cut, but this cutting may be required. In one embodiment, these linkers are not removed during processing.

The type of linker that can be present in the chimeric protein of the present disclosure is a protease cleavable linker that contains a cleavage site (ie, a protease cleavage site substrate, such as factor XIa, Xa, or thrombin cleavage site) And it may include additional linkers on the N-terminus or C-terminus or on both sides of the cleavage site. These cleavable linkers, when incorporated into the constructs of the present disclosure, produce chimeric molecules with heterologous cleavage sites.

In one embodiment, the FVIII polypeptide encoded by the nucleic acid molecule of the present disclosure comprises two or more Fc domains or portions, which are connected in a single polypeptide chain via a cscFc linker to form an Fc region . The cscFc linker is flanked by at least one intracellular processing site, that is, a site that is cleaved by an intracellular enzyme. Cleavage of the polypeptide at the at least one intracellular processing site produces a polypeptide comprising at least two polypeptide chains.

Other peptide linkers can optionally be used in the constructs of the present disclosure, for example to link the FVIII protein to the Fc region. Some exemplary linkers that can be used in conjunction with the present disclosure include, for example, the GlySer amino acid-containing polypeptides described in more detail below.

In one embodiment, the peptide linker is synthetic, i.e. non-naturally occurring. In one embodiment, the peptide linker includes a peptide (or polypeptide) (which may or may not be naturally occurring) comprising an amino acid sequence that will not be naturally connected to it in nature or The genetically fused first linear amino acid sequence and the second linear amino acid sequence are linked or genetically fused. For example, in one embodiment, the peptide linker may comprise a non-naturally-occurring polypeptide, which is a modified form of a naturally-occurring polypeptide (eg, contains a mutation, such as an addition, a substitution, or a deletion). In another embodiment, the peptide linker may comprise a non-naturally occurring amino acid. In another embodiment, the peptide linker may include naturally-occurring amino acids that are present in linear sequences that do not exist in nature. In yet another embodiment, the peptide linker may comprise a naturally occurring polypeptide sequence.

For example, in certain embodiments, peptide linkers can be used to fuse the same Fc portion, thereby forming a homodimeric scFc region. In other embodiments, peptide linkers can be used to fuse different Fc parts (such as wild-type Fc parts and Fc part variants), thereby forming a heterodimeric scFc region.

In another embodiment, the peptide linker comprises or consists of the gly-ser linker. In one embodiment, the scFc or cscFc linker comprises at least a part of an immunoglobulin hinge and a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide composed of glycine and serine residues. In some embodiments, the gly-ser linker can be inserted between two other sequences of the peptide linker. In other embodiments, the gly-ser linker is connected to one or both ends of another sequence of the peptide linker. In yet other embodiments, two or more gly-ser linkers are incorporated in series in a peptide linker. In one embodiment, the peptide linker of the present disclosure comprises at least a part of the upper hinge region (for example, derived from IgG1, IgG2, IgG3 or IgG4 molecules), at least a part of the middle hinge region (for example, derived from IgG1, IgG2, IgG3 or IgG4 molecule) and a series of gly/ser amino acid residues.

The length of the peptide linker of the present disclosure is at least one amino acid and can have varying lengths. In one embodiment, the peptide linker of the present disclosure has a length of about 1 to about 50 amino acids. As used in this context, the term "about" indicates +/- two amino acid residues. Since the length of the linker must be a positive integer, a length of about 1 to about 50 amino acids means a length of 1-3 to 48-52 amino acids. In another embodiment, the peptide linker of the present disclosure has a length of about 10 to about 20 amino acids. In another embodiment, the peptide linker of the present disclosure has a length of about 15 to about 50 amino acids. In another embodiment, the peptide linker of the present disclosure has a length of about 20 to about 45 amino acids. In another embodiment, the peptide linker of the present disclosure is about 15 to about 35 or about 20 to about 30 amino acids in length. In another embodiment, the length of the peptide linker of the present disclosure is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or 2000 amine groups acid. In one embodiment, the peptide linker of the present disclosure has a length of 20 or 30 amino acids.

In some embodiments, the peptide linker may comprise at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or At least 100 amino acids. In other embodiments, the peptide linker may comprise at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 amino acids. In some embodiments, the peptide linker may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 , 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 amino acids. The peptide linker can contain 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amines Base acid, 200-300 amino acid, 300-400 amino acid, 400-500 amino acid, 500-600 amino acid, 600-700 amino acid, 700-800 amino acid , 800-900 amino acids or 900-1000 amino acids.

The peptide linker can be introduced into the polypeptide sequence using techniques known in the art. The modification can be confirmed by DNA sequence analysis. The plastid DNA can be used to transform host cells for stable production of the produced polypeptide.

B.3.m. Monomer-dimer hybrid

In some embodiments, the isolated nucleic acid molecule of the present disclosure further comprising a heterologous nucleotide sequence encodes a monomer-dimer hybrid molecule comprising FVIII.

The term "monomer-dimer hybrid" as used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain that are associated with each other through disulfide bonds, wherein, for example, the first chain comprises Factor VIII and a first Fc region, and the second chain includes a second Fc region without FVIII, consists essentially of the second Fc region, or consists of the second Fc region. Therefore, a monomer-dimer hybrid construct is a hybrid comprising a monomer aspect with only one coagulation factor and a dimer aspect with two Fc regions.

B.3.n. Performance control elements

In some embodiments, the nucleic acid molecule or vector of the present disclosure further comprises at least one performance control sequence. The performance control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, which facilitates efficient transcription and translation of the encoding nucleic acid to which it is operably linked. For example, the isolated nucleic acid molecule of the present disclosure can be operably linked to at least one transcription control sequence.

The gene expression control sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters of the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter and other constitutive promoters . Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, those derived from cytomegalovirus (CMV), simian virus (such as SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rouss The long terminal repeat (LTR) of sarcoma virus, cytomegalovirus, Moloney's leukemia virus and other retrovirus promoters, and the thymidine kinase promoter of herpes simplex virus.

Other constitutive promoters are known to those of ordinary skill in the industry. Promoters that can be used as gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of inducers. For example, the metallothionein promoter is induced in the presence of certain metal ions to promote transcription and translation. Other inducible promoters are known to those of ordinary skill in the industry.

In one embodiment, the present disclosure includes the performance of gene transfer under the control of tissue-specific promoters and/or enhancers. In another embodiment, a promoter or other performance control sequence selectively enhances the performance of gene transfer in hepatocytes. Examples of liver-specific promoters include, but are not limited to, mouse transthyretin promoter (mTTR), endogenous human factor VIII (F8) promoter, endogenous human factor IX (F9) promoter, human α-1- Antitrypsin promoter (hAAT), human albumin minimal promoter and mouse albumin promoter. In a specific embodiment, the promoter includes the mTTR promoter. The mTTR promoter is described in RH Costa et al., 1986, Mol. Cell. Biol. 6:4697. The F8 promoter is described in Figueiredo and Brownlee, 1995, J. Biol. Chem . 270:11828-11838. In certain embodiments, the promoter includes any one of mTTR promoters (for example, mTTR202 promoter, mTTR202opt promoter, mTTR482 promoter), as disclosed in the US Patent Publication No. US2019/0048362, and the document is passed The reference is incorporated herein in its entirety.

One or more enhancers can be used to further enhance the level of performance to achieve therapeutic efficacy. One or more enhancers can be provided alone or together with one or more promoter elements. Generally, the performance control sequence contains multiple enhancer elements and tissue-specific promoters. In one embodiment, the enhancer includes one or more copies of the α-1-microglobulin/bikunin enhancer (Rouet et al., 1992, J. Biol. Chem . 267:20765-20773 ; Rouet et al., 1995, Nucleic Acids Res. 23:395-404; Rouet et al., 1998, Biochem . J. 334:577-584; Ill et al., 1997, Blood Coagulation Fibrinolysis 8: S23-S30). In another embodiment, the enhancer is derived from liver-specific transcription factor binding sites, such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, and Enh1, including HNF1, (sense)-HNF3, (sense)- HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4 (antisense).

In a specific example, a promoter that can be used in the present disclosure includes SEQ ID NO: 63 (ie, the ET promoter), which is also known as GenBank number AY661265. See also Vigna et al., Molecular Therapy 11(5) :763 (2005). Examples of other suitable vectors and gene regulatory elements are described in the following documents: WO 02/092134, EP1395293 or US Patent Nos. 6,808,905, 7,745,179 or 7,179,903, which are incorporated herein by reference in their entirety.

Generally, performance control sequences should include 5'non-transcribed sequences and 5'non-translated sequences that are involved in the initiation of transcription and translation respectively, such as TATA box, capping sequence, CAAT sequence, etc., as needed. In particular, such 5'non-transcribed sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked encoding nucleic acid. The gene expression sequence optionally includes an enhancer sequence or an upstream promoter sequence as required. Instance

Example 1-Recombinant Lentiviral Vector (LV) Preparation

The stability of the lentiviral vector drug product is assessed by exposing the vector to various stress conditions (such as freezing and thawing (F/T), elevated temperature (37ºC), stirring) and monitoring changes over time. Methods to determine stability include: ddPCR for functional titer, p24 ELISA for p24 concentration, and NanoSight for particle size distribution and particle concentration. Functional titer is a cell-based assay (HEK293) in which LV is cultured with cells, integrated into the cell genome, extracted, and DNA is measured by ddPCR. The ELISA-based p24 method is a kit-based method (Invitrogen) in which the viral capsid protein p24 is measured and it is related to the total particle concentration. NanoSight is a method that uses Brownian motion of particles to evaluate the size and concentration of LV particles in a solution.

Functional titer is a dose-defining parameter and a key metric. It provides information on whether the LV is stable and can therefore integrate its payload into the cell (related to the efficacy and mechanism of action of the drug). Functional titer is the standard for defining dose.

Vector generation and measurement

As described in the OXB patent (US 9,169,491 B2), the VSV pseudotyped third-generation lentiviral vector (LV) is generated by transiently co-transfecting four plastids into HEK293T cells, and purified by anion exchange . The carrier particles were initially analyzed by functional titer and HIV-I gag p24 antigen immunocapture (NEN Life Science Products) to ensure proper transfection, production and purification yields. For all vectors, the range of the expression titer or functional titer of the concentrated vector is 1-10E8 TU/mL transduction unit ^293T (TU)/ml.

Cell culture

The functional titer was measured by transducing adherent HEK293T cells with lentiviral vectors. The cells were divided three times and then harvested for genomic DNA isolation. LV integration is measured in genomic DNA by droplet digital PCR (ddPCR) using lentiviral vector-specific primers and probes. HEK293T adherent cells were maintained in Iscove's modified Dulbecco's medium (IMDM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and a combination of penicillin-streptomycin and glutamic acid.

Process LV into vehicle (preparation)

After purification, the LV material (API-DS) is combined, and the hollow fiber membrane is used to exchange its buffer into the corresponding preparation buffer. The DS is first concentrated approximately 10 times, and then exchanged with the corresponding formulation buffer six times the volume of the concentrated DS (for example , 6 mL buffer is used for every 1 mL of concentrated DS). The final formulated LV was considered a drug product (DP), and its stability was tested. Figures 7A to 7B and Figures 8A to 8B show the use of the vehicle TSSM (20 mM Tris, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3) After processing into vehicle phosphate ( formulation 1 ) (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3), the Characterization. In addition to TSSM, four alternative formulations were tested in this article: Formulation 2 (higher phosphate salt). 10 mM phosphate, 130 mM NaCl, 1% (w/v) sucrose, 0.05% (w/ v) P188, pH 7.3; Formulation 3 (histidine). 20 mM histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 6.5; Formulation 4 (Phosphate pH 7.0). 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.0; Formulation 5 (histidine pH 7.0). 20 mM Histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.0.

Example 2-In vivo administration of lentiviral vector (LV) preparations

5-week-old CD-1 and C57BL6 mice were purchased from Charles Rivers Laboratories and maintained under specific pathogen-free conditions. Six HemA male mice were obtained from the colony we raised in Charles River. The administration of vehicle or vehicle alone is carried out by tail vein injection or temporal vein injection in mice. All animal procedures were performed according to the protocol approved by Bioverativ/Sanofi IACUC (animal protocol 547). Three different formulations were tested: (1) 20 mM Tris, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3 (TSSM); (2) 10 mM phosphoric acid Salt, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3 (phosphate); and (3) 20 mM histidine, 100 mM NaCl, 3% (w/v) v) Sucrose, 0.05% (w/v) P188, pH 6.5 (histidine) (Table 4).

Table 4: In vivo administration of LV preparations Formulation handle mortality rate Observation results No reaction TSSM Carrier + vehicle x (n=1) x [Rough fur, excessive grooming, arched back posture] (n=5) TSSM Separate vehicle x [Rough fur, excessive grooming, arched back posture] (n=4) Phosphate Carrier + vehicle x (n = 3) Phosphate Separate vehicle x (n = 3) Histidine Carrier + vehicle x (n = 3) Histidine Separate vehicle x (n = 3)

Formulation: Vehicle alone (TSSM) and vehicle (TSSM) and LV

After a single intravenous administration, LV-coFIX was used for dose-response studies in C57BL6 and CD-1 adult mice. Eleven male C57BL6 mice (5 weeks old) and 11 male CD-1 mice (5 weeks old) were used. The doses administered are as follows: 6E10 (n = 3), 2E10 (n = 4) and 7.5E9 (n = 4) TU/kg. The formulation vehicle is TSSM buffer.

The LV-FIX vector formulated in TSSM buffer was used to administer C57BL6 and CD-1 mice. The 6E10 TU/kg dose is directly administered at 13.3-15 ml/kg without dilution. Before administration, the other two lower doses were diluted in PBS. Three mice of each strain were given high doses. Immediately after the injection of 6E10 TU/kg, a C57BL6 mouse suffered cardiac arrest and died. About half an hour later, the other mice in the group exhibited adverse effects. We observed excessive grooming, and then observed rough fur, lethargy, and inactivity. These mice appeared to recover one hour after the injection. Only the mice in the high dose (6E10 TU/kg) group showed adverse effects. The mice that received the diluted dose showed no adverse effects at all. Later, 2 CD-1 mice and 2 C57BL6 mice were administered only TSSM vehicle formulation buffer at 10-15 ml/kg. These mice also exhibited adverse effects (overgrooming, then rough fur, lethargy, and inactivity) after the injection, which normalized after about an hour. The results are summarized in Table 4.

Formulation: separate vehicle (phosphate)

HemA mice were used for dose-response studies. Three male HemA mice (9 weeks old) were used. The dose administered is 15 ml/kg. The formulation vehicle is phosphate buffered saline.

Three HemA mice were administered with 15 ml/kg phosphate buffer to test for any adverse effects in vivo. Observe the mice closely for the next 2 days. Did not notice any adverse effects, including excessive grooming, rough fur, lethargy, and inactivity seen when using TSSM to prepare the buffer.

Formulation: vehicle (phosphate) and LV

LV-coFVIII-6XTEN was used for dose-response studies in HemA mouse pups by temporal intravenous injection. 22 male and female HemA pups (2 days old) were used. The doses administered are as follows: 3E9 and 1.5E9 TU/kg. The formulation vehicle is phosphate buffered saline.

LV-FVIIIXTEN formulated in phosphate buffered saline was administered to two-day-old mice at 3E9 or 1.5E9 TU/kg through temporal intravenous injection. Give high doses without dilution (3E9). After injection, no adverse effects were seen in these mice.

Formulation: separate vehicle (histidine)

HemA mice were used for dose-response studies. Three male HemA mice (19 weeks old) were used. The dose administered is 15 ml/kg. The formulation vehicle is histidine buffer.

Three HemA mice were administered with 15 ml/kg histidine buffer to test for any adverse effects in vivo. Observe the mice closely for the next 2 days. Did not notice any adverse effects, including excessive grooming, rough fur, lethargy, and inactivity seen when using TSSM to prepare the buffer.

Formulation: vehicle (histidine) and LV

A dose-response study was performed using LV-coFVIII-6XTEN in tolerant adult HemA mice by tail vein injection. Four male HF8 mice (12 weeks old) were used. The dose administered is 15 ml/kg. The formulation vehicle is histidine buffer.

Four tolerant HemA mice (HF8) were administered with 15 ml/kg LV-FVIIIXTEN carrier formulated in histidine preparation buffer. No adverse effects were noted, including excessive grooming, rough fur, lethargy, and inactivity seen when using the TSSM formulation.

In summary, for each formulation, mice were injected with a vehicle or a separate vehicle. Table 4 shows that TSSM injection (both vehicle and vehicle alone) had adverse toxic effects on mice, including one death. In contrast, phosphate and histidine formulations (vehicle or vehicle alone) did not produce a response (no toxic effect) in mice.

Example 3-Testing of formulations using the vehicle TSSM

Stirring, freezing-thawing and temperature conditions

Test with or without addition of 1% (w/v) P188 (final concentration of 1%) by subjecting the formulation to stirring, freeze-thaw (F/T) cycles (5 and 10), and 6 hours room temperature incubation. w/v) Stability of the TSSM formulation (carrier vehicle) of P188) ( Figure 3A to Figure 3B , Figure 4 , Figure 5A to Figure 5B , Table 5 ). The stability of the carrier is measured by measuring functional titer, p24 concentration, and particle size and distribution (NanoSight). Figure 3A-3B, 4, 5A to 5B and Table 5, for the formulation with or without the P188, under different conditions, no significant change in stability of the vector. Use NanoSight to determine carrier integrity through particle size measurement.

Table 5: Testing of formulations using vehicle TSSM     ddPCR NanoSight p24 NanoSight/ddPCR p24/ddPCR Formulation stress TU/mL Particles /mL Particles /mL Infection ratio Infection ratio TSSM To 1.33E+08 6.08E+11 9.46E+11 4571 7109 TSSM + P188 To 1.27E+08 6.74E+11 1.00E+12 5292 7876 TSSM Stir 1.45E+08 6.50E+11 1.03E+12 4471 7101 TSSM + P188 Stir 1.45E+08 6.46E+11 1.21E+12 4443 8339 TSSM RT- 6h 1.29E+08 NT 1.04E+12 NT 8047 TSSM + P188 RT- 6h 1.34E+08 NT 1.13E+12 NT 8461 TSSM F/T 1.49E+08 6.18E+11 8.81E+11 4153 5919 TSSM + P188 F/T 1.72E+08 6.78E+11 9.66E+11 3945 5620 *Assuming 1 ng/mL p24 = 1.25E7 particles/mL (NT = not tested)

Dilution conditions

The TSSM formulation (vehicle plus vehicle) was diluted 1, 20, and 100 times, incubated at 37ºC, and stability was determined by measuring functional titers via ddPCR on days 0, 3, 7 and 14. Figures 6A and 6B show that dilution has no effect on stability for two weeks at elevated temperatures.

Continuous training for different periods of time

Incubate the TSSM formulation (vehicle) at 37ºC for 0, 3, 7 or 14 days. Stability is measured by determining functional titer via ddPCR, while particle integrity (particle concentration) is measured using Nanosight or p24 ELISA. Determining the time of culture with the stability and integrity of the carrier (particle concentration), as shown in FIGS. 7A-7B and 8A-8B. As the incubation time increases, the stability of the carrier decreases and the particle concentration increases.

Extended culture at 37ºC

Incubate the TSSM formulation (vehicle) at 37ºC for 0 days, 3 days, 1 week, or 2 weeks. Use Nanosight to measure particle size distribution. As shown in FIGS. 7A-7B and 8A to 8B, the incubation time for the particle size distribution becomes broad, while increasing particle concentration. The results can be explained as follows. The total particle concentration is increasing, while the infectivity is decreasing. This is because long-term exposure to 37ºC will rupture the capsid, which makes the p24 measured in ELISA more obvious. In addition, the rupture of the virus leads to an increase in smaller-sized substances.

Example 4-Test formulations using vehicle phosphate and histidine

Phosphate formulation

After processing into the vehicle phosphate (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3), the lentiviral vector (LV) formulation Characterize. Use Nanosight to measure particle size and distribution ( Figure 1 ). Figure 1 is a graph showing the results of Nanosight, which shows the clean monomer peak of the bulk drug (DS) cell after ultrafiltration/diafiltration into the final vehicle buffer (tangential flow filtration-TFF After), the peak drifted slightly to a larger particle size and showed the presence of some larger particles. Without being bound by theory, this may be due to the physical deterioration of the particles during the stress of the processing material. When filtered through a 0.22 µm filter membrane, the final drug product (DP) produces a particle size and distribution curve that reverts to the same size as the DS pool at the beginning of the treatment. The TFF stress is visually shown in the photos of FIGS. 2A to 2B.

The carrier stability of the phosphate formulation (vehicle vehicle) was tested under different conditions including stirring, incubation at 37ºC, duration and dilution ( Figure 9 and Figures 10A to 10B ).

Figures 10A to 10B show the NanoSight size data in phosphate buffer at 37ºC for one week. During the incubation time, the higher molecular weight substances increased slightly, and the total particles decreased overall. The data indicates that the loss of functional titer shown in Figure 9 may correspond to the physical loss of particles observed in Figures 10A to 10B during stability at RT or 37ºC.

In order to evaluate the stability of the phosphate formulation during a typical infusion scenario during clinical administration, a simulated in-use study was conducted. The LVV material is diluted with a phosphate vehicle and injected into an empty IV bag. Collect data within 6 hours at room temperature ( Figure 13 ). Stability studies show no signs of loss based on functional titer or p24, which indicates that the vector is stable for the duration of the study.

In order to test the compatibility with the prospective container closure system, a study was conducted to check the stability of the carrier and phosphate vehicle in Schott type 1 glass vials and West CZ COP vials ( Figure 14 , Figure 15, and Figure 16A to Figure 16C) ). The data showed that when only the vehicle was treated in a type 1 or CZ vial, no obvious particles were produced ( Figure 14 ). To evaluate the strain on the vial during freezing and thawing, the strain gauge was glued to the glass vial and placed in a -80ºC freezer for a period of 2 hours, followed by rapid heating in a 37ºC water bath ( Figure 15 ). Studies have shown that there is no perceivable normal strain on the vial, which would indicate that the phosphate formulation does not apply high stress to the container at this relatively high filling volume of 5 mL. The literature has shown that high stress can damage some Vial. The compatibility of the LVV material with each container was also tested, as shown in Figure 16A to Figure 16C . Through each test, namely transduction titer ( Figure 16A ), p24 ( Figure 16B ) and particle concentration ( Figure 16C ), the carrier has comparable stability and integrity, which shows that it is compatible with the two container closed forms (ie glass vials). Compatibility with plastic vials).

Comparison of phosphate and histidine formulations

The carrier stability of the phosphate (formulation 1 and formulation 2 ) and histidine ( formulation 3 ) formulations (carrier vehicle) was tested under different stress conditions (Figure 11A to Figure 11B , Figure 12A to Figure 12B ). In the freeze-thaw cycle study, it was observed that the functional titers of both phosphate formulations decreased as the cycle progressed. In contrast, the histidine formulation showed no loss of function and remained stable. In the process of maintaining room temperature and stirring stress conditions, the phosphate formulation showed a high level of functional loss (down to the limit of quantification). Surprisingly, the histidine formulation is still not affected by incubation for 3 days or stirring for 3 days at room temperature. In addition, for phosphate formulations (compare formulation 1 and formulation 2 ), adding more sodium chloride and reducing sucrose did not affect carrier stability.

In a separate formulation of the material, the retest of the stability study shown in Figures 11A to 11B was repeated and reported in Figure 17 . The loss of functional titer is not as severe as the phosphate formulation, but the trend between the histidine formulation and the phosphate formulation is consistent.

During the 9-month freezing (-80ºC) stability study, both phosphate formulation 1 and histidine formulation 3 buffers seemed to enable stable LVV DP ( Figure 18 ). Based on the functional titer, the data indicate that there is no material loss (within the measurement variability) during storage at -80ºC.

Finally, in order to evaluate the effects of only buffers (phosphate and histidine), two formulations at the same pH 7.0 were prepared in the same way. That is, the only difference between Formulation 4 and Formulation 5 is the buffer component (phosphate or histidine). In the context of the manufacturing process, the two formulations performed similarly in ultrafiltration and diafiltration (TFF) and final sterile filtration ( Table 6 ).

surface 6 :make Preparation of formulations by tangential flow filtration 4 (Phosphate) and formulations 5 (Histidine). The sample was diafiltered against six volumes of the corresponding formulation and then ultrafiltered (concentrated) several volumes. Formulation Process steps Volume (mL) NanoSight (Particles/mL) Total particles Phosphate Before TFF 165.2 1.28E+11 2.11E+13 Phosphate 0.2 um after filtering 88.5 2.11E+11 1.87E+13 Histidine Before TFF 155.8 9.64E+10 1.50E+13 Histidine 0.2 um after filtration 65.2 2.54E+11 1.66E+13

After preparing the corresponding formulations, stability studies were performed to compare buffer components. Figures 19A to 19D summarize the findings shown in functional titers and normalized functional titers, p24 and particle concentration as a function of stability conditions: there is only a slight difference between the LVV stability of either formulation. This seems to indicate that the results presented in Figure 11A to Figure 11B , Figure 12A to Figure 12B, and Figure 13 seem to reflect changes due to differences in pH (7.3 and 6.5) rather than buffer composition (phosphate or histidine) The level of stability.

none

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings. The patent or application file contains at least one drawing in color. After requesting and paying the necessary fees, the official will provide a copy of this patent or patent application publication with one or more color drawings.

Figure 1 depicts the treatment of lentiviral vector (LV) into vehicle phosphate (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3). ) Characterization of the formulation. The bulk drug (DS) cell showed a clean monomer peak. After ultrafiltration/diafiltration into the final vehicle buffer (after tangential flow filtration-TFF), the peak drifted slightly to a larger size. And there are some larger particles. Without being bound by theory, this may be due to the physical deterioration of the particles during the stress of the processing material. The final DP has been filtered through a 0.22 µm filter membrane, and the distribution curve is restored to coincide with the DS pool at the beginning of the treatment. The influence of TFF stress can be seen visually in the pictures shown in Figs. 2A to 2B.

Figures 2A to 2B depict the sterile filtration pair after tangential flow filtration (TFF) in the vehicle phosphate (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) The effect of lentiviral vector (LV) in P188, pH 7.3) ( Figure 2A ) and in the final drug substance (DS) pool (Figure 2B).

Figures 3A to 3B depict the lentiviral vector (LV) when 1% (w/v) P188 is not added ( Figure 3A ) and when 1% (w/v) P188 is added ( Figure 3B) Stability in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3), as measured by p24 concentration using p24 ELISA.

Figure 4 depicts the lentiviral vector (LV) in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3) while stirring The stability, as measured by particle concentration and particle size using NanoSight. Stability studies using TSSM formulations: 20 mM Tris, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3, such as those with and without Poloxomer 188 (P188) Circumstances indicated. Use NanoSight for measurement.

Figures 5A to 5B depict the lentiviral vector (LV) in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol, pH 7.3) The stability in the measurement, as measured by particle concentration and particle size using NanoSight. Figure 5A shows that under agitation stress, the size of the lentiviral particles seems to only increase slightly. It is assumed that the main peak is the monomer lentiviral vector (about 130 nm), and the smaller and larger peak may be the degradation of the monomer particle. Figure 5B shows that the addition of 1% (w/v) poloxamer 188 (P188) does not interfere with the lentiviral particles.

Figures 6A to 6B depict the lentiviral vector (LV) in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1%) at 37ºC at 0, 3, 7 and 14 days (W/v) Mannitol, pH 7.3), as measured by functional titer in TU/ml (Figure 6A ) and T0% ( Figure 6B) using ddPCR. Figure 6B is normalized to time 0. Each bar group represents the following dilution series: undiluted (no dilution), 20-fold dilution (20x), and 100-fold dilution (100x).

Figures 7A to 7B depict the lentiviral vector (LV) in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1%) at 37ºC at 0, 3, 7 and 14 days (W/v) Mannitol, pH 7.3), such as using ddPCR through functional titer (in TU/ml) and using NanoSight through particle concentration ( Figure 7A ) or through functional titer superimposed with p24 data ( Figure 7B ) Measured.

Figure 8A to Figure 8B . Figure 8A depicts the lentiviral vector (LV) at 37ºC in the vehicle TSSM (20 mM TRIS, 100 mM NaCl, 1% (w/v) sucrose, 1% (w/v) mannitol) , PH 7.3) changes with the culture time (in days and weeks), as measured by the particle concentration and size using Nanosight. Figure 8B reports the results of the 37ºC stability experiment on a logarithmic scale to more clearly see the difference in particle size over time.

Figure 9 depicts the lentiviral vector (LV) in the vehicle phosphate formulation (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3) Incubation time, incubation temperature, stirring and freezing/thawing (F/T) cycles (in days), as measured by functional titer (in T0%) using ddPCR and p24 concentration using p24 ELISA .

Figures 10A to 10B depict a lentiviral vector (LV) using a phosphate formulation (10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3) Stability at 37ºC over time (in days), as measured by particle concentration and particle size distribution using NanoSight. Figure 10A reports the NanoSight data normalized to 1 to make the difference in the degradation peaks more obvious. Figure 10B presents the original data, which shows the decrease of the monomer peak over time at 37ºC.

Figures 11A to 11B depict the stability of the lentiviral vector (LV), which compares three formulations: Formulation 1. Phosphate formulation: 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose , 0.05% (w / v) P188, pH 7.3; formulation 2 .10 mM phosphate, 130 mM NaCl, 1% ( w / v) sucrose, 0.05% (w / v) P188, pH 7.3; formulation 3 . 20 mM histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 6.5; during 5 and 10 freezing and thawing (F/T) cycles ( Figure 11A ), and at room temperature (RT) and at room temperature and stirring (orbital shaker, 350 rpm) for 3 days ( Figure 11B ), as measured by functional titer using ddPCR.

Figures 12A to 12B depict the stability of lentiviral vectors (LV), which compares three formulations: formulation 1 (Phos). Phosphate formulation: 10 mM phosphate, 100 mM NaCl, 3% (w/ v) Sucrose, 0.05% (w/v) P188, pH 7.3; Formulation 2 (Phos. higher salt). 10 mM phosphate, 130 mM NaCl, 1% (w/v) sucrose, 0.05% (w/ v) P188, pH 7.3; Formulation 3 (Hist). 20 mM histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 6.5, if NanoSight is used to pass particles Quantity measured. Figure 12A shows the data at room temperature (RT) and at room temperature and stirring (orbital shaker, 350 rpm) for 3 days, while Figure 12B shows the freezing and thawing (F/ T) Circular data.

Figure 13 depicts a simulated in-use stability study using one of the following formulations during exposure to an IV bag at room temperature for 6 hours: Formulation 1 (phosphate buffer): 10 mM phosphate, 100 mM NaCl, 3% ( w/v) Sucrose, 0.05% (w/v) P188, pH 7.3.

Figure 14 depicts a formulation buffer stability study comparing the performance of container closures (Schott type 1 glass vials and West CZ COP vials) in a freezing and thawing cycle (-80ºC overnight and thawing in a 37ºC water bath): Preparation Substance 1 (phosphate buffer): 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3. Use microfluidic imaging (MFI) to obtain particle concentration.

Figure 15 depicts a vial strain study using a Schott type 1 glass vial after a freezing and thawing cycle (-80C, thawed in a 37ºC water bath): Formulation 1 (phosphate buffer only): 10 mM phosphate, 100 mM NaCl , 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3. Use strain gauges and thermocouples to collect data.

Figures 16A to 16C depict LVV material compatibility, which compares the performance of container closures (Schott type 1 glass vials and West CZ vials) after various stability conditions, 1FT = 1 freezing and thawing cycle, 2 hr = exposure 2 hr at room temperature; 3x foam is an aggressive suction and dispensing from the pipette to produce visible foam in the container; 10x is the same stability parameter at a 10-fold dilution. LVV is in Formulation 1 (phosphate buffer): 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3; such as by functional titer ( Figure 16A ), p24 concentration (Figure 16B ) and particle concentration ( Figure 16C ) were measured. Use strain gauges and thermocouples to collect data. Use NanoSight to obtain particle concentration.

Figure 17 depicts a stability study comparing two formulations after 10 freezing and thawing (F/T) cycles and comparing 1 day and 3 days at room temperature (RT): Formulation 1 ( phosphoric acid Salt buffer): 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3; Formulation 3 (histidine buffer). 20 mM histamine Acid, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 6.5.

Figure 18 depicts the long-term stability of phosphate and histidine formulations (9 months of frozen storage at -80ºC (9 mo.)) data : Formulation 1 (phosphate): 10 mM phosphate, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) P188, pH 7.3; Formulation 3 (histidine). 20 mM histidine, 100 mM NaCl, 3% (w/v) Sucrose, 0.05% (w/v) P188, pH 6.5.

Figures 19A to 19D depict the stability study comparing phosphate buffer and histidine buffer at the same pH 7.0: Formulation 4 (phosphate): 10 mM phosphate, 100 mM NaCl, 3% (w/ v) Sucrose, 0.05% (w/v) P188, pH 7.0; Formulation 5 (histidine). 20 mM histidine, 100 mM NaCl, 3% (w/v) sucrose, 0.05% (w/v) ) P188, pH 7.0; as measured by functional titer ( Figure 19A ), normalized functional titer ( Figure 19B ), p24 concentration ( Figure 19C ) and particle concentration ( Figure 19D ). The results are shown in units of functional titer and normalized to the percentage of starting material (TO). Use NanoSight to obtain particle concentration.

Claims (48)

A recombinant lentiviral vector preparation, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) No TRIS buffer system; (c) Salt; (d) Surfactants; and (e) carbohydrates, The pharmaceutical composition is suitable for systemic administration to human patients. The formulation of claim 1, wherein the buffer system or the pH of the formulation is about 6.0 to about 8.0. The formulation of claim 1 or 2, wherein the buffer system or the pH of the formulation is about 6.0 to about 7.5. The formulation according to any one of the preceding claims, wherein the buffer system or the pH of the formulation is about 6.0 to about 7.0. The formulation according to any one of the preceding claims, wherein the buffer system or the pH of the formulation is about 6.5. The formulation according to any one of the preceding claims, wherein the buffer system or the pH of the formulation is about 7.0 to about 8.0. The formulation according to any one of the preceding claims, wherein the buffer system or the pH of the formulation is about 7.3. The preparation according to any one of the preceding claims, wherein the lentiviral vector comprises a nucleotide sequence encoding VSV-G or a fragment thereof. The formulation according to any one of the preceding claims, wherein the buffer system comprises phosphate buffer or histidine buffer. The formulation according to claim 9, wherein the concentration of the phosphate buffer is about 5 mM to about 30 mM. The formulation according to claim 10, wherein the concentration of the phosphate buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM , Or about 15 mM to about 20 mM. The formulation according to claim 9, wherein the concentration of the histidine buffer is about 5 mM to about 30 mM. The formulation according to claim 12, wherein the concentration of the histidine buffer is about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, or about 15 mM to about 20 mM. The formulation according to any one of the preceding claims, wherein the concentration of the salt is about 80 mM to about 150 mM. The formulation of any one of the preceding claims, wherein the concentration of the salt is about 100 mM, about 110 mM, about 130 mM, or about 150 mM. The formulation according to any one of the preceding claims, wherein the salt is a chloride salt. The formulation according to claim 16, wherein the chloride salt is NaCl. The formulation according to any one of the preceding claims, wherein the surfactant is poloxamer. The formulation of claim 18, wherein the poloxamer is selected from the group consisting of poloxamer 101 (P101), poloxamer 105 (P105), poloxamer 108 (P108) ), Poloxamer 122 (P122), Poloxamer 123 (P123), Poloxamer 124 (P124), Poloxamer 181 (P181), Poloxamer 182 (P182), Poloxamer Poloxamer 183 (P183), Poloxamer 184 (P184), Poloxamer 185 (P185), Poloxamer 188 (P188), Poloxamer 212 (P212), Poloxamer 215 (P215) , Poloxamer 217 (P217), Poloxamer 231 (P231), Poloxamer 234 (P234), Poloxamer 235 (P235), Poloxamer 237 (P237), Poloxamer 238 (P238), Poloxamer 282 (P282), Poloxamer 284 (P284), Poloxamer 288 (P288), Poloxamer 331 (P331), Poloxamer 333 (P333), Poloxamer 334 (P334), Poloxamer 335 (P335), Poloxamer 338 (P338), Poloxamer 401 (P401), Poloxamer 402 (P402), Poloxamer 403 (P403), Poloxamer 407 (P407) and their combinations. The formulation according to claim 18 or 19, wherein the poloxamer is poloxamer 188 (P188). The formulation according to claim 18 or 19, wherein the poloxamer is poloxamer 407 (P407). The formulation according to any one of claims 1-17, wherein the surfactant is polysorbate. The formulation of claim 22, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof . The formulation according to any one of the preceding claims, wherein the concentration of the surfactant is about 0.01% (w/v) to about 0.1% (w/v). The formulation according to any one of the preceding claims, wherein the concentration of the surfactant is about 0.03% (w/v), about 0.05% (w/v), about 0.07% (w/v), or about 0.09% (W/v). The formulation of any one of the preceding claims, wherein the concentration of the carbohydrate is about 0.5% (w/v) to about 5% (w/v). The formulation according to any one of the preceding claims, wherein the concentration of the carbohydrate is about 1% (w/v), about 2% (w/v), about 3% (w/v), or about 4% (w/v). %(W/v). The formulation according to any one of the preceding claims, wherein the carbohydrate is sucrose. The formulation according to any one of claims 1-28, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) About 10 mM phosphate; (c) About 100 mM sodium chloride; (d) About 0.05% (w/v) Poloxamer 188; and (e) about 3% (w/v) sucrose, Wherein the pH of the formulation is about 7.3, and where the pharmaceutical composition is suitable for systemic administration to human patients. The formulation according to any one of claims 1-28, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) About 10 mM phosphate; (c) About 130 mM sodium chloride; (d) About 0.05% (w/v) Poloxamer 188; and (e) about 1% (w/v) sucrose, Wherein the pH of the formulation is about 7.3, and where the pharmaceutical composition is suitable for systemic administration to human patients. The formulation according to any one of claims 1-28, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) About 20 mM histidine; (c) About 100 mM sodium chloride; (d) About 0.05% (w/v) Poloxamer 188; and (e) about 3% (w/v) sucrose, Wherein the pH of the formulation is about 6.5, and where the pharmaceutical composition is suitable for systemic administration to human patients. The formulation according to any one of claims 1-28, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) About 10 mM phosphate; (c) About 100 mM sodium chloride; (d) About 0.05% (w/v) Poloxamer 188; and (e) about 3% (w/v) sucrose, Wherein the pH of the formulation is about 7.0, and where the pharmaceutical composition is suitable for systemic administration to human patients. The formulation according to any one of claims 1-28, which comprises: (a) A therapeutically effective dose of recombinant lentiviral vector; (b) About 20 mM histidine; (c) About 100 mM sodium chloride; (d) About 0.05% (w/v) Poloxamer 188; and (e) about 3% (w/v) sucrose, Wherein the pH of the formulation is about 7.0, and where the pharmaceutical composition is suitable for systemic administration to human patients. The preparation according to any one of the preceding claims, wherein the recombinant lentiviral vector comprises a nucleic acid comprising at least the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A nucleotide sequence that is 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. The preparation according to claim 34, wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The preparation according to claim 34 or 35, wherein the recombinant lentiviral vector comprises a nucleic acid composed of the factor VIII (FVIII) coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The preparation according to any one of claims 1-36, wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises at least 80%, at least at least 80% of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3 85%, at least 90%, at least 95%, or at least 99% identical nucleotide sequences. The preparation according to claim 37, wherein the recombinant lentiviral vector comprises a nucleic acid, and the nucleic acid comprises the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. The preparation according to claim 37 or 38, wherein the recombinant lentiviral vector comprises a nucleic acid composed of the factor IX (FIX) coding sequence shown in SEQ ID NO: 3. The formulation according to any one of the preceding claims, wherein the recombinant lentiviral vector comprises an enhanced transthyretin (ET) promoter. The preparation according to any one of the preceding claims, wherein the recombinant lentiviral vector further comprises a nucleotide sequence that is at least 90% identical to the target sequence of miR-142 shown in SEQ ID NO: 7. The preparation according to any one of the preceding claims, wherein the recombinant lentiviral vector is isolated from a transfected host cell selected from the group consisting of CHO cells, HEK293 cells, BHK21 cells, PER.C6 cells, NSO cells, and CAP cell. The preparation according to claim 42, wherein the host cell is a CD47-positive host cell. A method for treating a human patient suffering from a disorder, which comprises administering the recombinant lentiviral vector preparation according to any one of the preceding claims to the human patient. The method of claim 44, wherein the formulation is administered systemically to the human patient. The method according to claim 44 or 45, wherein the formulation is administered intravenously. The method of any one of claims 44-46, wherein the disorder is a bleeding disorder. The method according to claim 47, wherein the bleeding disorder is hemophilia A or hemophilia B.

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